Phenylbenzopyrones structure-activity studies identify betuletol derivatives as potential antitumoral agents

Article abstract of DOI:10.1016/j.ejphar.2006.07.020

We have analyzed the cytotoxicity of 22 compounds with a phenylbenzo-γ-pirone core structure, most of them obtained from natural sources, in five human tumor cell lines (HL-60, A431, SK-OV-3, HeLa and HOS). Betuletol 3-methyl ether and its diacetate were

Full text of DOI:10.1016/j.ejphar.2006.07.020

European Journal of Pharmacology 548 (2006) 9–20
www.elsevier.com/locate/ejphar
Phenylbenzopyrones structure-activity studies identify betuletol derivatives
as potential antitumoral agents
Sara Rubio a, José Quintana a, Mariana López b, José Luis Eiroa b, Jorge Triana b,
Francisco Estévez a,
⁎
a
Department of Biochemistry, I.C.I.C., University of Las Palmas de Gran Canaria, Plaza Dr. Pasteur s/n, 35016 Las Palmas de Gran Canaria, Spain
b
Department of Chemistry, I.C.I.C., University of Las Palmas de Gran Canaria, Campus Universitario de Tafira, 35017 Las Palmas de Gran Canaria, Spain
Received 10 March 2006; received in revised form 20 June 2006; accepted 13 July 2006
Available online 22 July 2006
Abstract
We have analyzed the cytotoxicity of 22 compounds with a phenylbenzo-γ-pirone core structure, most of them obtained from natural sources,
in five human tumor cell lines (HL-60, A431, SK-OV-3, HeLa and HOS). Betuletol 3-methyl ether and its diacetate were the most cytotoxic
compounds. The HL-60 cell line was especially sensitive to these compounds, with IC50 values of approximately 1 μM. Treatment of HL-60 cells
with betuletol 3-methyl ether was associated with apoptosis induction which was prevented by a non-specific caspase inhibitor (z-VAD-fmk) and
also by a specific inhibitor of caspase-8 (z-IETD-fmk) indicating activation of the extrinsic apoptotic pathway. The results suggest that betuletol 3-
methyl ether has potential as new anticancer agent.
© 2006 Elsevier B.V. All rights reserved.
Keywords: Apoptosis; Flavonoids; Cytotoxic activity; Caspases; DNA fragmentation; poly(ADP-ribose) polymerase
1. Introduction
activation of caspase-3 (Li et al., 1997; Srinivasula et al., 1998).
The extrinsic pathway is initiated by activation of death
receptors and thereby cleavage of procaspase-8 (Boldin et al.,
1996; Muzio et al., 1996). Caspase-8 directly activates caspase-
3 (Stennicke et al., 1998). Caspase-8 also cleaves Bid-a Bcl-2
family member— and thereby plays a role in the release of
cytochrome c. However, it is becoming increasingly clear that
apoptosis can also occur independently of caspase activation
(Chipuk and Green, 2005).
Flavonoids comprise a vast array of biologically active
compounds ubiquitous in plants, many of which have been used
in traditional Eastern medicine for thousands of years. These
polyphenolic compounds exert several biological functions such
as apoptosis-inducing activity, free radical scavenging activity,
and anti-tumorigenic activity (Ko et al., 2002; Lee et al., 2002).
Here we have studied the effect of twenty two flavonoids, most
of them isolated from endemic plants to the Canary Islands, on
cell viability of five human tumor cell lines. The betuletol
derivatives, betuletol 3-methyl ether and betuletol 3-methyl
ether diacetate were the most potent cytotoxic compounds in all
cell lines studied, but also the most potent apoptotic inducers on
Many anticancer drugs have been shown to cause the death
of sensitive cells through the induction of apoptosis. This
cellular suicide program is initiated by ligation of death
receptors, growth factor deprivation or various environmental
stresses which are mediated by molecular pathways that
culminate in the activation of a conserved family of aspartate-
specific cysteine proteases, known as the caspases, which
orchestrate the dismantling and clearance of the dying cell.
Caspases themselves are present as proenzymes that are readily
cleaved and activated during apoptosis, providing the cell with a
means to rapidly amplify its apoptotic response (Cohen, 1997;
Thornberry and Lazebnik, 1998). Two major pathways have
been identified for the induction of apoptosis. The intrinsic
pathway is activated by release of mitochondrial cytochrome c
(Kluck et al., 1997; Liu et al., 1996; Yang et al., 1997) which
forms a complex with Apaf-1 and activates caspase-9 leading to
⁎
Corresponding author. Tel.: +34 928 451443; fax: +34 928 451441.
E-mail address: festevez@dbbf.ulpgc.es (F. Estévez).
0014-2999/$ - see front matter © 2006 Elsevier B.V. All rights reserved.
doi:10.1016/j.ejphar.2006.07.020

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S. Rubio et al. / European Journal of Pharmacology 548 (2006) 9–20
human myeloid leukemia HL-60 cells. Increased levels of anti-
apoptotic or decreased levels of pro-apoptotic proteins can cause
acquired or intrinsic resistance against apoptosis induction by
antineoplastic drugs thereby contributing to treatment failure
and poor clinical prognosis. Thus, novel agents targeting
aberrant apoptosis pathways or inducing alternative death
pathways may be suited to overcome treatment resistance
(Igney and Krammer, 2002). The present studies demonstrate
that betuletol 3-methyl ether induces apoptosis, at least in large
part, by activation of the extrinsic caspase-8 pathway of death.
5,7-diacetoxy-4′-hydroxy-3,6-dimethoxyflavone 14: UV/
Vis λmax nm, (MeOH): 287, 323,329; (+NaOMe): 284, 325,
393; (+AlCl3): same as MeOH; (+AlCl3+ HCl): same as AlCl3;
(+NaOAc): 287, 360; (+NaOAc+H3BO3): 287,325. PMR
(300 MHz, CDCl3): δ 8.06 (d, J=8.8, H-2′, H-6′), 7.21 (d,
J=8.5, H-3′, H-5′), 6.89 (s, H-8), 3.94 (s, OMe), 3.76 (s, OMe),
2.50 (s, OAc), 2.33 (s, OAc). EIMS m/z (rel. int.): 413 [M]+
(36.6), 372 (100), 330 (49.2), 315 (46.8), 312 (42.8), 287 (22.5),
269 (10.6), 121 (14.9), 69 (15.6).
Quercetin 4 (3,3′,4′,5,7-pentahydroxyflavone) was obtained
from Sigma. Cryptostrobin 18 was isolated from Rumohra
adiantiformis. The structure of this compound was determined
by a combination of spectroscopic analysis and comparison
with reported data (Byrne et al., 1982).
2. Materials and methods
2.1. Materials
Dihydroquercetin 7,3′-dimethyl ether 16 and dihydrokaemp-
ferol 7,4′-dimethyl ether 17 were isolated from the aerial parts
of Pulicaria canariensis as recently described (Triana et al.,
2005). All other reagents were of analytical grade. Purity of all
compounds was 99.0% as judged by high-performance liquid
chromatography. Stock solutions of 10 mM flavonoids were
made in dimethyl sulfoxide (DMSO), and aliquots were frozen
at −20 °C. Polyvinylidene difluoride (PVDF) membranes were
purchased from Millipore. Antibodies for poly(ADP-ribose)
polymerase (PARP), caspase-3, caspase-8 and caspase-9 were
purchased from Stressgen. Antibody for cytochrome c was
purchased from BD PharMingen. Secondary antibodies were
from Amersham Pharmacia Biotech. The caspase inhibitors
were from Sigma. The caspase-3 and caspase-8 substrates were
from Sigma. The caspase-9 substrate was from AnaSpec.
Most of the flavonoids used in this study were obtained from
the Canary Islands' endemic plants and were isolated according
to published methods with minor modifications. Structural
identities of flavonoids were determined spectroscopically
(proton nuclear magnetic resonance and 13C nuclear magnetic
resonance, infrared and UV/Visible spectroscopy and mass
spectrometry) as described previously: Betuletol 3-methyl ether
1, quercetin 3,3′-dimethyl ether 7, kaempferol 3-methyl ether 9,
5,7-dihydroxy-3,3′,4′-trimethoxyflavone 10, 4′, 5, 7-trihy-
droxy-3,6-dimethoxyflavone 12 and eriodictyol 19 were
isolated from the aerial parts of Allagopappus dichotomus as
described (González et al., 1995). Quercetin 3-methyl ether 5,
naringenin 21, eriodictyol 19 and kaempferol 3-methyl ether 9
were isolated from Allagopappus viscosissimus as described
(González et al., 1992a). Axillarin 11 and apigenin 22 were
isolated from the aerial parts of Tanacetum ferulaceum and Ta-
nacetum ptarmiciflorumas described (González et al., 1990,
1992b). Acetyl derivatives of betuletol 3-methyl ether (com-
pound 2), quercetin 3-methyl ether (compound 6), quercetin
3,3′-dimethyl ether (compound 8) and eriodictyol (compound
20) were obtained by treatment of the corresponding alcohols
with acetic anhydride (Ac2O) in pyridine for 12 h at room
temperature. Betuletol 3, 5, 7-trimethyl ether 3 was obtained by
methylation of 4′,5,7-trihydroxy-3, 6-dimethoxyflavone 12.
Mono-di- and triacetyl derivatives (compounds 13, 14 and 15)
of 4′,5,7-trihydroxy-3, 6-dimethoxyflavone 12 were obtained by
acetylation of this compound with acetic anhydride in pyridine
as above. The acetyl derivatives were purified by chromatog-
raphy on a silica gel column, eluted with hexane, and hexane-
ethyl acetate mixtures (8:2). The mono and diacetyl derivatives,
7-Acetoxy-4′,5-dihydroxy-3,6-dimethoxyflavone 13 and 5,7-
diacetoxy-4′-hydroxy-3,6-dimethoxyflavone 14, are described
for the first time.
2.2. Cell culture
HL-60 cells were cultured in RPMI 1640 medium containing
10% (v/v) heat-inactivated fetal bovine serum and 100 units/ml
penicillin and 100 μg/ml streptomycin at 37 °C in a humidified
atmosphere containing 5% CO2. The cultures were passed twice
weekly exhibiting characteristic doubling times of ∼24 h. The cell
numbers were counted by a hematocytometer, and the viability was
always greater that 95% in all experiments as assayed by the
0.025% trypan blue exclusion method. Further dilutions of stock
solutions of flavonoids were made in culture media just before use.
In all experiments, the final concentration of DMSO did not exceed
0.3% (v/v), a concentration which is non-toxic to the cells. The
same concentration was present in control experiments. The A431
cell line was grown as monolayers in plastic tissue flasks containing
Dulbecco's modified Eagle's medium (DMEM) supplemented
with 10% fetal bovine serum, 100 units/ml penicillin and 100 μg/ml
streptomycin. SK-OV-3 was grown as monolayers in DMEM. The
human cervix carcinoma HeLa and human osteosarcoma HOS
cells were grown in Eagle's minimum essential medium (EMEM)
supplemented with 2 mM glutamine, 1% Non essential amino acids
and 10% fetal bovine serum. Cells at logarithmic growth phase
were harvested using 0.25% trypsin-EDTA solution for 5 min,
pelleted by centrifugation at 500 ×g for 5 min, washed once with
medium and resuspended at a concentration of 1×104 cells/ml.
These cell lines were obtained from the European Collection of Cell
Cultures (Salisbury, UK).
7-Acetoxy-4′,5-dihydroxy-3,6-dimethoxyflavone 13: UV/
Vis λmax nm, (MeOH): 289, 316,330; (+NaOMe): 286, 325,
398; (+AlCl3): 294, 350, 396; (+AlCl3+ HCl): same as AlCl3;
(+NaOAc): 290, 376; (+NaOAc+H3BO3): same as NaOAc.
PMR (300 MHz, CDCl3): δ 8.11 (d, J=8.8 Hz, H-2′, H-6′),
7.23 (d, J=8.8 Hz, H-3′, H-5′), 6.76 (s, H-8), 4.04 (s, OMe),
3.87 (s, OMe), 2.35 (s, OAc). EIMS m/z (rel. int.): 372 [M]+
(100), 357 (15.6), 330 (41.1), 329 (45.8), 287 (39.1), 269 (17.8),
121 (22.9), 69 (66.0).

S. Rubio et al. / European Journal of Pharmacology 548 (2006) 9–20
11
Human peripheral blood mononuclear cells (PBMC) were
isolated from heparin-anticoagulated blood of healthy volunteers
by centrifugation with Ficoll–Paque Plus (Amersham Bios-
ciences). PBMCs were also stimulated with phytohemagglutinine
(PHA, 2 μg/ml) for 48 h before experimental treatment.
containing 50 μg/ml propidium iodide and 100 μg/ml RNase A
and incubation for 1 h at 37 °C in the dark. The percentage of
cells with decreased DNA staining, composed of apoptotic cells
resulting from either fragmentation or decreased chromatin, of a
minimum of 10,000 cells per experimental condition was
counted. Cell debris was excluded from analysis by selective
gating based on anterior and right angle scattering.
2.3. Cytotoxicity of flavonoids on HL-60 cells, A431 cells,
HeLa cells, SK-OV-3 cells and HOS cells
2.6. Analysis of DNA fragmentation
The cytotoxicity of flavonoids in HL-60 cells was analyzed
by colorimetric 3-(4,5-dimethyl-2-thiazolyl-)-2,5-diphenyl-2H-
tetrazolium bromide (MTT) assay. Briefly, 1×104 exponential-
ly growing cells were seeded in 96-well microculture plates
with various flavonoids concentrations. After the addition of
MTT (0.5 mg/ml) cells were incubated at 37 °C for 4 h. Sodium
dodecyl sulfate (SDS) (10% w/v) in 0.05 M HCl was added to
the wells and then incubated at room temperature overnight
under dark conditions. The absorbance was measured at
570 nm. Concentrations inducing a 50% inhibition of cell
growth (IC50) were determined graphically for each experiment
as described (Rivero et al., 2003). A431, SK-OV-3, HeLa and
HOS cells (5×103 −1×104) were inoculated in 96-well plates
and were then incubated at 37 °C for 24 h to allow the
attachment to plates after which the culture media were changed
and flavonoids were added and provided different final
concentrations. Cells were then incubated at 37 °C in a 5%
CO2–95% air atmosphere for 72 h. Cell viability was
determined as above using a colorimetric assay with the
reduction of MTT.
HL-60 cells were washed with PBS and incubated in 20 μl of
50 mM Tris-HCl (pH 8.0), 10 mM EDTA, 0.5% SDS and 1 μg/
μl RNase A (Sigma) at 37 °C for 1 h. Then, 10 μg/μl proteinase
K (Sigma) (2 μl) was added, and the mixture was incubated at
50 °C for 2 h more. DNA was extracted with 100 μl of phenol-
chloroform-isoamyl alcohol (24:24:1) and mixed with 5 μl of
loading solution (10 mM EDTA, pH 8.0, containing 1% (w/v)
low melting-point agarose, 0.25% bromophenol blue and 40%
sucrose). Samples were separated by electrophoresis in 2%
agarose gels and visualized by UV illumination after ethidium
bromide staining.
2.7. Immunoblotting of caspase-3
HL-60 cells (1×106) were treated with flavonoids at the
indicated concentrations in RPMI 1640 medium. Cell pellets
were lysed in lysis buffer containing 125 mM Tris-HCl pH 6.8,
2% SDS, 5% glycerol, and 1% β-mercaptoethanol, and boiled
for 5 min. The samples were separated on 15% SDS-
polyacrylamide gel, electrotransferred to a PVDF membrane,
immunoblotted with anti-caspase-3 antibody [Stressgen; 1:1000
dilution in Tris-buffered saline containing 0.1% Tween-20
(TBST) supplemented with 3% nonfat milk] overnight. After
washing and incubation with horseradish peroxidase-conjugat-
ed anti-rabbit (Amersham Pharmacia Biotech), the antigen–
antibody complexes were visualized by enhanced chemilumi-
nescence (ECL, Amersham Pharmacia Biotech) using the
manufacturer's protocol.
2.4. Quantitative fluorescent microscopy
Cells were harvested and fixed in 3% paraformaldehyde and
incubated at room temperature for 10 min. The fixative was
removed and the cells were washed with PBS, resuspended in 30–
50 μl of PBS containing 20 μg/ml bis-benzimide trihydrochloride
(Hoechst 33258), and incubated at room temperature for 15 min.
Ten-microliter aliquots of the cells were placed on glass slides,
and triplicate samples of 500 cells each were counted and scored
for the incidence of apoptotic chromatin condensation using a
Zeiss fluorescent microscopy. Stained nuclei with condensed
chromatin (supercondensed chromatin at the nuclear periphery),
or nuclei that were fragmented into multiple smaller dense bodies
were considered as apoptotic. Nuclei with uncondensed and
dispersed chromatin were considered as not apoptotic.
2.8. Immunoblot analysis of poly(ADP-ribose) polymerase
degradation
Induction of apoptosis was also examined by proteolytic
cleavage of poly(ADP-ribose) polymerase. After treatments, cells
were pelleted by centrifugation, and resuspended in lysis buffer
containing 25 mM PBS, 0.1 mM phenylmethylsufonylfluoride
and protease inhibitors leupeptin, aprotinin and pepstatin A (5 μg/
ml each). After centrifugation, the pellet was resuspended in the
loading buffer containing 125 mM Tris-HCl, pH 6.8, 2% SDS,
5% glycerol, and 1% β-mercaptoethanol. The mixture was
sonicated for 30 s at 4 °C and then boiled to 100 °C for 3 min. The
cell lysates were fractionated on a 7.5% polyacrylamide gel
containing 0.1% SDS and proteins were electrophoretically
transferred onto a PVDF membrane. Membrane was blocked with
5% nonfat milk in Tris-buffered saline containing 0.1% Tween-20
for 1 h, followed by incubation with anti-poly(ADP-ribose)
polymerase polyclonal antibody (Stressgen; 1:3000 dilution in
2.5. Quantification of apoptosis by flow cytometry
To study changes in the cell DNA content, histogram
measurements of hypodiploid DNA formation was performed
by flow cytometry using a Coulter EPICS™ cytometer (Beck-
man Coulter). Histograms were analyzed with the Expo 32 ADC
Software™. Cells were collected and centrifuged at 500 ×g,
washed with PBS and resuspended in 50 μl of PBS. Following
dropwise addition of 1 ml of ice-cold 75% ethanol, fixed cells
were stored at −20 °C for 1 h. Samples were then centrifuged at
500 ×g and washed with PBS before resuspension in 1 ml of PBS

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S. Rubio et al. / European Journal of Pharmacology 548 (2006) 9–20
TBST supplemented with 3% nonfat milk) overnight. After
washing and incubation with anti-rabbit antibody conjugated to
horseradish peroxidase (Amersham Pharmacia Biotech), the
antigen–antibody complexes were visualized by enhanced
chemiluminiscence (ECL, Amersham Pharmacia Biotech) using
the manufacturer's protocol. The appearance of an 85 kDa
cleavage product was used as a measure of apoptosis.
Antiproliferative studies on betuletol 3-methyl ether 1 indicate
that this compound displays strong cytotoxic properties in all
cell lines assayed, although HL-60 cells was especially sensitive
to this substance with an IC50 as low as 1.8 0.9 μM (Table 1).
Since betuletol 3-methyl ether 1 has two hydroxyl groups on C5
and C7, respectively, we next obtained the diacetate 2- and
dimethyl ether 3-derivatives of betuletol 3-methyl ether 1 in
order to know more about the structural requirement for the
antiproliferative action of this compound. Therefore, the
cytotoxic studies performed with the di-acetylated compound
indicate that theses changes have no impact on the IC50 in HL-60
cells (1.8 0.9 μM vs 1.3 0.4 μM). However, in the
dimethylated derivative a dramatic consequence on the growth
inhibitory activity is observed. Contrary to betuletol 3-methyl
ether 1 and betuletol 3-methyl ether diacetate 2, betuletol 3, 5, 7-
trimethyl ether 3 is ineffective as an antiproliferative agent in all
cell lines tested. Since the methoxy group is smaller than the
acetyl group, it seems clear that this is not a consequence of steric
hindrance. Moreover, if the hydroxyl groups in the betuletol 3-
methyl ether 1 interact by hydrogen bridges with vicinity groups
on the biological targets, then the presence of the keto groups on
the di-acetyl derivative could be important. One group that
seems to play a key role in determining the potency of these
compounds on cell viability is the methyl group on position 4′of
the B ring (2-phenyl group) since betuletol 3-methyl ether 1 is
significantly more potent than 4′,5,7-trihydroxy-3,6-dimethox-
yflavone 12 in all cell lines studied.
The critical relationship of fruit and vegetable intake and
cancer prevention has been thoroughly documented (Block et al.,
1992). Since quercetin 4 is the most abundant flavonoid in the
human diet we have investigated the effect of a series of
structurally related flavonoids. The results indicate that methyl-
ation of hydroxyl group at position C3 of quercetin 4 yields
quercetin 3-methyl ether 5, a compound with a higher
antiproliferative activity. Cytotoxic effects were observed in all
cancer cell lines tested except in HOS cells, which indicates that
the above chemical change in the quercetin molecule 4 affects cell
proliferation in a cell-type specific manner. However, the
hydroxyl group located at the 3′ position seems to have little
importance for the growth inhibitory activity of the above
quercetin derivative since the additional methylation at this level
to yield quercetin 3,3′-dimethyl ether 7 does not significantly
change the IC50 value. The presence of a hydroxyl group in the 3′
position of ring B has different consequences depending on the
configuration of molecule. For example, kaempferol 3-methyl
ether 9 lacks the 3′-hydroxyl group and displays similar IC50 to its
3′-hydroxylated counterpart, quercetin 3-methyl ether 5 in all cell
lines assayed. Similarly, quercetin 3-methyl ether tetracetate 6 and
quercetin 3,3′-dimethyl ether triacetate 8 differ only by the
presence of an acetyl- or methylether-group, respectively, at the 3′
position, but they showed similar potency. On the other hand, the
presence of the 3′ hydroxyl group improves the antiproliferative
activity of axillarin 11 as compared with the 3′-dehydroxylated
counterpart 12 in all cell types tested except HL-60 cells. When a
methoxy group is placed at C6 instead of hydrogen in quercetin 3-
methyl ether 5, the impact on the IC50 is dependent on the cell type
under study. The resulting compound (axillarin 11) is significantly
2.9. Detection of cytochrome c release from mitochondria
Release of cytochrome c from mitochondria was detected by
Western blot analysis. After treatments, HL-60 cells were
washed twice with PBS and then suspended in ice-cold buffer
[20 mM HEPES (pH 7.5), 1.5 mM MgCl2, 10 mM KCl, 1 mM
EDTA, 1 mM EGTA, 1 mM dithiothreitol, 0.1 mM phenyl-
methylsulfonylfluoride, and 5 μg/ml leupeptin, aprotinin, and
pepstatin A] containing 250 mM sucrose. After 15 min
incubation on ice, cells were lysed by pushing them several
times through a 22-gauge needle and the lysate spun down at
1000 ×g for 5 min at 4 °C. The supernatant fraction was
centrifuged at 105,000×g for 45 min at 4 °C and the resulting
supernatant was used as the soluble cytosolic fraction. Cytosolic
proteins (50 μg) were resolved on an SDS/15% polyacrylamide
gel and electrotransferred onto a PVDF membrane. The
membrane was probed with monoclonal anti-cytochrome c
antibody (BD Transduction Laboratories) (1:250 dilution) and
then with secondary antibody conjugated to horseradish
peroxidase. Proteins bands were detected by chemiluminescence
(ECL, Amersham Pharmacia Biotech) as described above.
2.10. Assay of caspase activity
After treatments, cells were harvested by centrifugation at
1000 ×g for 5 min at 4 °C and washed with PBS, and the cell
pellets were kept on ice. The cells were resuspended in cell lysis
buffer (50 mM HEPES, pH 7.4, 1 mM dithiothreitol, 0.1 mM
EDTA, 0.1% Chaps) and held on ice for 5 min. After
centrifugation for 10 min at 17,000 ×g at 4 °C, the supernatants
were analyzed for protein concentration by the Bradford dye-
binding assay and stored at −20 °C until used to study caspase
colorimetric enzymatic activity. Equal amounts of protein
(∼20 μg) from different treatments were used, and the assays
were set up on ice. The net increase of absorbance at 405 nm
after incubation at 37 °C was indicative of enzyme activity.
Specific labelled substrates for caspase-3, -8 and 9 activities
were N-acetyl-Asp-Glu-Val-Asp-p-nitroaniline (DEVD-
pNA), N-acetyl-Ile-Glu-Thr-Asp-p-nitroaniline (IETD-pNA)
and N-acetyl-Leu-Glu-His-Asp-p-nitroaniline (LEHD-pNA)
respectively.
3. Results
3.1. Betuletol derivatives inhibit the growth and cell viability of
human tumor cell lines
In the present study, we examined the effects of 22 flavonoids
(Fig. 1), on the growth of five human tumor cell lines.

S. Rubio et al. / European Journal of Pharmacology 548 (2006) 9–20
13
Fig. 1. Structures of the investigated flavonoids.
less cytotoxic on HL-60 (IC50=14.3 4.6 μM versus 32.6
8.8 μM) but significantly more cytotoxic on HOS cells
(IC50=90.4 27.6 μM versus 25.6 9.6 μM). In contrast, the
presence of this methyl ether group does not change its cytotoxic
potency on A431, HeLa and SK-OV-3 cells.
(90.4 27.6 μM versus 9.4 3.0). Unlike the HOS cells, the
remaining cell lines were resistant to these chemical changes.
Also, an increase in the growth inhibitory activity was clearly
observed when quercetin 3,3′-dimethyl ether 7 was acetylated at
positions 4′, 5 and 7 to produce quercetin 3,3′-dimethyl ether
triacetate 8. HL-60 cells, A431 cells and particularly HOS cells
were susceptible to these chemicals substitutions.
Selected acetylations were also performed on the free
hydroxyl groups of the 4′, 5, 7-trihydroxy-3, 6-dimethoxy-
flavone molecule 12. Acetylation of the hydroxyl group at
position 7 yielded a monoacetylated derivative, 7-acetoxy-4′,5-
dihydroxy-3,6-dimethoxyflavone 13, that was significantly
more active on HL-60 cells. This effect is abolished when a
second and a third acetyl group are sequentially introduced at
position 5 and at position 4′ to produce the derivative
compounds 5,7-diacetoxy-4′-hydroxy-3,6-dimethoxyflavone
14 and 4′,5,7-triacetoxy-3,6-dimethoxyflavone 15 respectively.
Another set of flavonoids are those related to the structure of
the low cytotoxic compound naringenin 21. This flavanone
decreased the proliferation rate of the cell lines studied only at
The influence of the hydroxyl group at position 4′ of the B ring
(2-phenyl group) on cell viability was also tested. A dramatic
decrease in the growth inhibitory activity was observed when
quercetin 3,3′-dimethyl ether 7 was methylated in that position to
produce 5,7-dihydroxy-3,3′,4′-trimethoxyflavone 10. The IC50
increased from 12.2 0.4 μM and 30.6 7.3 μM to N100 μM, in
HL-60 and HeLa cells respectively. As occurs with others
substitutions, this chemical change affected the cell lines under
study to different degrees. The HOS cell line was resistant to both
quercetin 3,3′-dimethyl ether 7 and 5,7-dihydroxy-3,3′,4′-
trimethoxyflavone 10. Thus, acetylation of all free hydroxyl
groups locating at positions 3′, 4′, 5 and 7 in the quercetin 3-
methyl ether 5 molecule to produce quercetin 3-methyl ether
tetracetate 6 greatly improved the cytotoxic activity on HOS cells.
In this cell line the IC50 decreased about one order of magnitude

14
S. Rubio et al. / European Journal of Pharmacology 548 (2006) 9–20
Table 1
3.2. Betuletol derivatives induce apoptosis on human myeloid
leukemia cells
Effects of phenylbenzo-γ-pirone derivatives on the growth of human tumor cell
lines
Compound IC50 (μM)
In order to evaluate whether betuletol derivatives decrease cell
viability through apoptosis activation, morphological changes
characteristic of apoptotic cells (condensed and fragmented
chromatin) were analyzed and quantified by fluorescent micros-
copy (Fig. 2A and B). Evaluation of nuclear morphology (Fig.
2B), indicates that the percentage of apoptotic cells increased
from 6 1% to 24 2% (4 times increase) in betuletol 3-methyl
ether treated cells, after 12 h exposure at a concentration as low as
10 μM (Fig. 2A). Next, we examined whether these flavonoids
induce poly(ADP-ribose) polymerase cleavage, a hallmark of
apoptosis that indicates activation of caspase. Hydrolysis of the
116 kDa poly(ADP-ribose) polymerase protein to the 85 kDa
fragment was detected in betuletol 3-methyl ether and betuletol 3-
methyl ether diacetate treated cells after 12 h exposure at 10 μM
(Fig. 2C), and increased in a dose-dependent manner (data not
shown). These results indicate that poly(ADP-ribose) polymerase
cleavage was involved in apoptosis induced by these compounds
and, as expected (due to cytotoxic ineffectiveness), betuletol
3,5,7-trimethyl ether did not induce poly(ADP-ribose) polymer-
ase cleavage (Fig. 2C). When cells were incubated with these
HL-60
A431
HeLa
SK-OV-3
HOS
1
2
3
1.8 0.9
1.3 0.4
N100
3.0 1.0
8.0 2.9
N100
9.1 4.0
8.1 4.7
N100
21.0 7.0
30.0 3.0
N100
6.8 0.6
7.5 0.8
N100
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
27.8 4.1
14.3 4.6
9.2 3.4
12.2 0.4
3.8 0.2
7.2 2.9
N100
32.6 8.8
27.5 1.6
13.4 3.1
33.5 0.3
20.6 3.1
N100
71.1 7.1
26.8 7.9
19.3 8.2
33.9 6.0
10.0 5.2
40.6 7.9
54.2 3.7
33.0 9.2
59.6 3.6
58.6 2.5
55.9 1.0
56.1 7.6
90.3 0.1
N100
N100
N100
93.3 1.1
35.0 11.7 44.6 11.9 90.4 27.6
24.7 3.6
30.6 7.3
29.3 5.7
43.6 15.3
30.3 12.3 N100
29.6 6.6 18.3 3.3
9.4 3.0
32.8 11.3 30.0 11.3 77.5 4.5
N100
47.7 8.8
N100
N100
N100
N100
N100
N100
68.0 41.0 N100
29.5 11.5 25.6 9.6
88.2 1.8
N100
N100
69.8 5.2
N100
N100
N100
N100
60.5 14.6
60.0 1.0
80.3 28.0
49.8 25.0
75.6 13.9
N100
91.7 12.3
N100
N100
N100
73.5 10.1 71.2 10.1 N100
35.0 3.0
4.5 1.3
N100
28.8 5.8
10.8 1.3
N100
N100
20.5 6.1
N100
22.5 2.9
N100
N100
82.6 15.3
13.1 1.0
76.3 18.9 32.7 12.4 71.3 6.1
Cells were cultured for 72 h and the IC50 values were calculated as described in
the Experimental Section. The data shown represent the mean S.E.M. of 3–5
independent experiments with three determinations in each.
very high concentrations (IC50 N100 μM) but the introduction
of a hydroxyl group at position 3′ (eriodictyol 19) greatly
increases its antiproliferative activity although only on HL-60
and A431 cells. The 3′, 4′, 5 and 7 tetracetylated derivative,
eriodictyol tetracetate 20, is even more active with IC50 values
as low as 4.5 1.3 μM and 10.8 1.3 μM on HL-60 and A431,
respectively. A moderate increase in the cytotoxicity of
naringenin 21 is also observed on HL-60 and A431 cells
when the hydroxyl group placed at the 4′ position on the B ring
(2-phenyl group) is absent and the molecule has a methyl group
on C8 (cryptostrobin 18). Another structural requirement to
improve the cytotoxic activity of naringenin 21 is the
introduction of a double bond between C2 and C3. The
resulting flavone (apigenin 22) showed antiproliferative activity
in all cell lines tested although HL-60 cell line was the most
susceptible to this chemical change (IC50 =13.1 1.0 μM).
A detailed inspection of Table 1 reveals that among the five
tumor cell lines used in this study the growth of promyelocytic
HL-60 cells were, in general, most susceptible to the cytotoxicity
induced by flavonoids. Interestingly, quercetin 4, a flavonoid
widely studied due to abundance in the fruit and vegetable
usually consumed by humans, displayed significantly cytotoxic
properties for HL-60 cells alone (IC50 =27.8 4.1 μM). The
remaining lines were insensitive to quercetin 4 effects on cell
proliferation.
Fig. 2. Effects of betuletol 3-methyl ether and betuletol 3-methyl ether diacetate
on apoptosis on HL-60 cells. (A) Cells were stained with bisbenzimide
trihydrochloride after treatment with 10 μM of betuletol 3-methyl ether for 12 h,
and apoptotic cells were determined by fluorescence microscopy. Values
represent means S.E. This histogram is representative of two independent
⁎
experiments each performed in duplicate. Pb0.05, significantly different from
the untreated control. (B) Photomicrographs of representative fields of HL-60
cells treated as above and stained with bisbenzimide trihydrochloride to evaluate
nuclear chromatin condensation (i.e. apoptosis). (C) Analysis for the cleavage of
poly(ADP-ribose) polymerase (PARP) by immunoblotting. (D) Cells were
incubated as above and DNA extracted. Laddered electrophoretic patterns of
oligonucleosomal DNA fragments were resolved by conventional agarose gel
electrophoresis, stained with ethidium bromide and visualized under UV light.
Control lane refers to untreated cells.
Since HL-60 cell line was highly sensitive to the anti-
proliferative effect of betuletol 3-methyl ether 1 we decided to
determine whether this natural flavonoid displays its cytotoxic
action through activation of the apoptotic pathway.

S. Rubio et al. / European Journal of Pharmacology 548 (2006) 9–20
15
flavonoids, the DNA showed the typical fragmentation patterns
formed by internucleosomal hydrolysis of chromatin thus
confirming the apoptosis-inducing effects. As expected, betuletol
3,5,7-trimethyl ether was unable to induce DNA fragmentation
(Fig. 2D). Taken together, these results indicate that both betuletol
derivatives, betuletol 3-methyl ether and betuletol 3-methyl ether
diacetate, are potent inducers of human myeloid leukemia HL-60
cells apoptosis and that both compounds are equally active.
3.4. Betuletol derivatives-induced apoptosis involves caspase-3
activation
To determine whether caspase-3 is activated in HL-60 cells by
the betuletol 3-methyl ether, dose-response experiments were
performed and cell lysates assayed for cleavage of the tetrapeptide
substrate N-acetyl-Asp-Glu-Val-Asp-p-nitroaniline (DEVD-
pNA). The results (Fig. 4A), demonstrate that caspase-3 activity
increased 1.5- and 2.3-fold in betuletol 3-methyl ether treated
cells with 3- or 10-μM of compound, respectively, for 12 h.
Hydrolysis of procaspase-3 to the active enzyme, caspase-3, by
betuletol 3-methyl ether and betuletol 3-methyl ether diacetate
was also determined by immunobloting. The results indicate that
these compounds at concentrations as low as 3 μM promote
cleavage of pro-caspase-3 into active caspase-3 (Fig. 4B). At high
concentrations (100 μM) of a specific caspase-3 inhibitor,
benzyloxycarbonyl-Asp-Glu-Val-Asp-(OMe) fluoromethyl ke-
tone (z-DEVD-fmk), the percentage of apoptotic cells and DNA
laddering induced by betuletol 3-methyl ether was significantly
decreased. In contrast, there was not any effect when the
concentration of the inhibitor used was lowered to a minimum
of 10 μM (results not shown).
3.3. A general caspase inhibitor abolished betuletol
derivatives-induced poly(ADP-ribose) polymerase cleavage
and DNA fragmentation
To confirm that betuletol derivatives-triggered apoptosis
requires the activation of caspases, HL-60 cells were pretreated
with the broad-spectrum caspase inhibitor z-VAD-fmk (benzy-
loxycarbonyl-Val-Ala-Asp(OMe) fluoromethyl ketone). The
results demonstrate that this inhibitor completely blocked
betuletol derivatives-induced DNA fragmentation (Fig. 3A).
The effect of z-VAD-fmk on poly(ADP-ribose) polymerase
cleavage by betuletol 3-methyl ether was also analyzed. As
observed (Fig. 3B), pretreatment of HL-60 cells with this
inhibitor completely abolished the generation of the 85 kDa
fragment. Taken together, these results indicate that these events
are caspase dependent.
3.5. Effects of betuletol derivatives on mitochondrial cytochrome
c release
To determine whether betuletol 3-methyl ether and betuletol
3-methyl ether diacetate-induced apoptosis on HL-60 cells
involves the release of cytochrome c from mitochondria to
cytosol, dose-response experiments were performed and cyto-
solic preparations were analyzed by immunoblotting. As
demonstrated (Fig. 5A), a low concentration of both compounds
(3 μM) induce cytochrome c release at the same proportion and
no difference was observed with higher concentrations of
compounds. The effect of these compounds on caspase-9
formation was also analyzed. The results indicate that both
betuletol derivatives stimulate cleavage of inactive pro-caspase-
9 to the active 37 kDa fragment (Fig. 5B). To determine whether
this fragment was associated with caspase-9 activity, lysates
from treated cells were assayed for cleavage of the specific
substrate N-acetyl-Leu-Glu-His-Asp-p-nitroaniline (LEHD-
pNA). The results indicated that compound 1 (10 μM, 12 h)
also increases caspase-9 activity (Fig. 5C). In order to determine
the contribution of caspase-9 to the betuletol derivatives-induced
apoptosis we examined the impact of the irreversible caspase-9
inhibitor benzyloxycarbonyl-Leu-Glu-His-Asp(OMe)-fluoro-
methyl ketone (z-LEHD-fmk). As shown (Fig. 5D) the inhibitor
was completely ineffective at blocking betuletol 3-methyl ether-
induced DNA nucleosomal fragmentation typical of apoptotic
cell death.
Fig. 3. Involvement of activation of caspases in the induction of apoptosis. HL-
60 cells were incubated with the indicated doses of compounds for 12 h in
absence or presence of broad-spectrum caspase inhibitor z-VAD-fmk (100 μM).
DNA fragmentation (A) and proteolysis of poly(ADP-ribose) polymerase (B)
were then analyzed. The migration positions of full-length poly(ADP-ribose)
polymerase and the 85 kDa cleavage product are indicated. The results are
representative of three independent experiments.
3.6. Caspase-8 is activated by betuletol derivatives
Since the intrinsic pathway does not seem to contribute
significantly to betuletol derivatives-induced apoptosis, we
decided to determine the contribution of the extrinsic pathway.
Therefore, HL-60 cells were treated with increasing doses of

16
S. Rubio et al. / European Journal of Pharmacology 548 (2006) 9–20
percentage of apoptotic cells from 6% (control) to 24% and 30%
with 10 μM and 30 μM of product, respectively, as determined
by flow cytometry (Fig. 6C). The effect of betuletol 3-methyl
ether was, however, completely abolished when the cells were
pretreated with z-IETD-fmk. No impact on basal apoptosis level
Fig. 4. Activation of caspase-3 in HL-60 cells. (A) The cells were incubated in
the presence of the indicated concentrations of betuletol 3-methyl ether for 12 h,
and total cell lysate was assayed for caspase-3 activity using the DEVD-pNA
colorimetric substrate. The results are expressed as fold-increase in caspase
activity compared with control. Values represent means S.E. This histogram is
representative of two independent experiments each performed in triplicate.
⁎
Pb0.05, significantly different from the untreated control. (B) Western blot
analysis for the cleavage of caspase-3. The cells were treated with different
concentrations of betuletol 3-methyl ether and betuletol 3-methyl ether diacetate
and whole cell lysates were subjected to sodium dodecyl sulfate-polyacrylamide
gel electrophoresis (SDS-PAGE) followed by blotting with an anti-caspase-3
antibody. β-Actin was used as loading control.
betuletol 3-methyl ether and the lysates were then assayed for
cleavage of the tetrapeptide N-acetyl-Ile-Glu-Thr-Asp-p-nitroani-
line (IETD-pNA) as a specific substrate for caspase-8. As shown
(Fig. 6A), this compound induced caspase-8 activation in a dose-
dependent manner. The enzymatic activity increased to 2.4- and
3.5-fold over control with 3 μM and 10 μM of betuletol 3-methyl
ether, respectively. Also, we subjected cell lysates to immunoblot
analysis with an antibody which recognizes the inactive
procaspase-8 and the proteolytic (32 kDa and 42–44 kDa)
fragments. The results (Fig. 6B), clearly demonstrate that
concentrations of betuletol 3-methyl ether as low as 3 μM
significantly promotes procaspase-8 hydrolysis. A dramatic
increase in the hydrolysis of procaspase-8 (visualized as 32 kDa
and 42–44 kDa fragments) was observed with the higher
concentration tested (10 μM) while the lowest concentration
used (1 μM) had no impact on the cleavage at the time assayed
(12 h). Proteolytic cleavage of the initiator caspase-8, which is
demonstrated in response to betuletol 3-methyl ether, typically
occurs after triggering cell surface death receptors like the CD95
receptor (Budihardjo et al., 1999; Krammer, 2000).
Fig. 5. Effects on the intrinsic pathway of apoptosis. (A) HL-60 cells were
incubated with the indicated concentrations of compounds 1 and 2 for 12 h,
cytosolic extracts were separated by sodium dodecyl sulfate-polyacrylamide gel
electrophoresis (SDS-PAGE) and cytochrome c release was detected by
immunoblotting. As loading control β-actin was also analyzed. (B) Western
blot analysis for the cleavage of procaspase-9. Cells were treated as above and
whole cell lysates were subjected to SDS-PAGE followed by immunoblotting
with an anti-procaspase-9 antibody that also recognizes the proteolytic
fragments generated. (C) Cells were incubated in the presence or absence of
betuletol 3-methyl ether for 12 h, and total cell lysates were assayed for caspase-
9 activity using the LEHD-pNA colorimetric substrate. The results are expressed
as fold-increase in caspase activity compared with control. Values represent
means S.E. This histogram is representative of two independent experiments
⁎
each performed in triplicate. Pb0.05, significantly different from the untreated
control. (D) Lack of effect of z-LEHD-fmk treatment on betuletol 3-methyl
ether-induced DNA fragmentation. Cells were preincubated in the presence or
absence of the caspase-9 inhibitor z-LEHD-fmk (100 μM) for 1 h and then
followed by treatment with betuletol 3-methyl ether for 12 h. DNA
fragmentation was analyzed by agarose gel electrophoresis as described in
Materials and methods.
To determine whether caspase-8 is implicated in the betuletol
3-methyl ether-induced apoptosis on HL-60 cells, we examined
the impact of the irreversible caspase-8 inhibitor benzylox-
ycarbonyl-Ile-Glu-Thr-Asp(OMe)-fluoromethyl ketone (z-
IETD-fmk). Therefore, betuletol 3-methyl ether increased the

S. Rubio et al. / European Journal of Pharmacology 548 (2006) 9–20
17
showed no similar effect. Low concentrations (10 μM) of each
specific caspase inhibitor (z-LEHD-fmk for caspase-9 and z-
IETD-fmk for caspase-8) were ineffective in decreasing the
percentage of apoptotic cells and on DNA laddering induced by
betuletol 3-methyl ether (results not shown).
To define the temporal relationships among the betuletol 3-
methyl ether-induced events, HL-60 cells were treated with this
compound and then harvested at various intervals. Induction of
both caspase-8 (3-fold) and caspase-3 (3.8-fold) activities was
significantly detectable at 8 h of treatment. By contrast, the
increase in caspase-9 activity (3-fold) was not detectable until
12 h (Fig. 7B). These findings indicated that betuletol 3-methyl
ether induces caspase-8 activation and thereby direct of
cleavage of caspase-3.
3.8. Lack of cytotoxicity of betuletol derivatives on normal
human lymphocytes
Since an ideal anti-cancer agent should have no effect on
normal, non-tumoral cells, we investigated whether betuletol
derivatives 1 and 2 were also cytotoxic for human peripheral
blood mononuclear cells (PBMC). No cytotoxicity (up 10 μM) to
either fresh or proliferating PBMC growth was observed. As a
Fig. 6. Activation of caspase-8. (A) HL-60 cells were treated with the indicated
concentrations of betuletol 3-methyl ether for 12 h and cell lysates were then
assayed for caspase-8 activity. (B) The cells were treated as described in (A) and
activation of caspase-8 was determined by immunoblot analysis. (C) HL-60
cells were incubated with the indicated concentrations of betuletol 3-methyl
ether for 12 h, in absence or presence of the caspase-8 inhibitor z-IETD-fmk
(100 μM). Apoptotic cells (i.e. hypodiploid DNA content) was determined and
quantified by flow cytometry after staining with propidium iodide. (D) Cells
were treated as above and genomic DNAwas extracted, separated on an agarose
gel and visualized under UV light by ethidium bromide staining. Values
represent means S.E. Histograms are representative of two independent
⁎
experiments each performed in triplicate. Pb0.05, significantly different
from the untreated control.
was detected in cells incubated with z-IETD-fmk. The effect of
caspase-8 inhibitor on DNA fragmentation induced by betuletol
3-methyl ether was also analyzed. As shown (Fig. 6D), in the
presence of z-IETD-fmk no DNA-laddering was observed in
HL-60 cells treated with 10 μM of betuletol 3-methyl ether.
These results demonstrate that caspase-8 plays a central role in
the caspase-dependent apoptosis induced by betuletol 3-methyl
ether.
3.7. Inhibition of caspase-3 activation in the presence of the
caspase-8 inhibitor but no effect by the caspase-9 inhibitor
Fig. 7. (A) Western blot analysis for the effects of z-IETD-fmk (caspase-
8 specific inhibitor) and z-LEHD-fmk (caspase-9 specific inhibitor) on betuletol
3-methyl ether-induced cleavage of procaspase-3 or poly(ADP-ribose) poly-
merase. (B) Kinetics of caspase-3, caspase-8 and caspase-9 activation in
response to betuletol 3-methyl ether. HL-60 cells were treated with 10 μM
betuletol 3-methyl ether and harvested at the indicated times. Cell lysates were
assayed for caspase-3, caspase-8 and caspase-9 activities. Results are expressed
as fold-increase in caspase activity compared with control. Values represent
means S.E. This histogram is representative of two independent experiments
To further improve the demonstration of the key role played
by caspase-8 in betuletol derivative-induced apoptosis, we
performed additional experiments.
Pretreatment of HL-60 cells with the irreversible caspase-
8 inhibitor z-IETD-fmk fully blocked the procaspase-3 and poly
(ADP-ribose) polymerase (a typical substrate for caspase-3)
cleavage (Fig. 7A), but the caspase-9 inhibitor z-LEHD-fmk
⁎
each performed in triplicate. Pb0.05, significantly different from the untreated
control.

18
S. Rubio et al. / European Journal of Pharmacology 548 (2006) 9–20
because betuletol 3-methyl ether 1 is much more potent than 4′,5,7-
trihydroxy-3,6-dimethoxyflavone 12 in all cell lines studied.
A study on structure-antiproliferative activity reported
relationships between several polymethoxylated flavonoids,
indicating that the C2–C3 double bond and the 3-hydroxyl
group of the C ring are important structural factors (Kawaii et al.,
1999). Our finding that naringenin 21 and dihydroquercetin 7,3′-
dimethyl ether 16 are not effective antiproliferative agents
confirmed that the C2–C3 double bond may be important. The
role of the 3′-hydroxyl group is deduced when comparing the
antiproliferative activities of naringenin 21 and eriodictyol 19.
The only structural difference in both flavonoids is the presence
of a 3′-hydroxyl group in the eriodictyol molecule 19, which
clearly increases its cytotoxic capabilities. These results must be
interpreted as cell-type specific since only HL-60 and A431 cells
were susceptible to eriodictyol 19.
Some authors have suggested that the growth-suppressive
activity of polymethoxylated flavonoids may, in part, be
attributed due to their chemical stability (Kandaswami et al.,
1993). It has been demonstrated that quercetin 4 may undergo
autoxidation and can also be oxidatively degraded, while
methylation of phenolic groups, as in the case of quercetin 3,3′-
dimethyl ether 7 would be expected to confer greater stability to
this flavonoid (Kandaswami et al., 1993). Moreover, addition of
ascorbic acid at low concentrations increases the antiprolifera-
tive activity of quercetin 4 against the HTB 43 squamous cell
carcinoma which seems to be related to the ability of ascorbic
acid to inhibit the oxidative degradation of the polyhydroxy-
lated flavonoids (Kandaswami et al., 1993). The finding of
significantly higher cytotoxic activity of quercetin 3,3′-
dimethyl ether 7 than quercetin 4 in most cell lines studied,
concords with this. Unlike quercetin 4, methylation of quercetin
3,3′-dimethyl ether 7 to yield quercetin 3,3′,4′-trimethyl ether
10 decreases its cytotoxic potency. Similarly, when betuletol 3-
methyl ether 1 is methylated to betuletol 3,5,7-trimethyl ether 3,
its cytotoxic activity is completely abolished. Although there
have been no studies on the chemical stability of betuletol and
its methoxylated derivatives, this result cannot be explained by
an increase in stability, but rather the loss of essential hydroxyl
group/s. Although there is much evidence that the position and
number of the hydroxyl groups on A and B rings strongly
influence the conformation of molecule and modulate their
growth inhibitory effect, it is generally accepted that the growth
inhibition effect of flavonoids cannot be predicted on the basis
of their chemical composition and structure (Kuntz et al., 1999).
However, the cytotoxicity of quercetin 4 was greatly improved
by acetylation and methylation of the free hydroxyl groups in all
cell lines tested. In HL-60 the cytotoxic potency increased ten
times. Since acetylated/methylated derivatives compounds can
pass through biological membranes more easily than quercetin, a
possible explanation of the difference in IC50 could be the higher
intracellular concentration reached. However, there are probably
additional factors that play important roles to determine the final
biological response in a particular tumor cell line. For example,
although the acetylated derivative of eriodictyol 20 greatly
improved the antiproliferative activity in most cell lines (HL-60,
A431, HeLa and SK-OV-3), the increase in lypophilicity does not
Fig. 8. Differential effect of betuletol derivatives on proliferation of normal
peripheral blood mononuclear cells (PBMC) vs. HL-60 cells. Proliferation of
HL-60 cells, quiescent PBMC and phytohemagglutinine (PHA)-activated
healthy human PBMC cultured in presence of indicated concentrations of
compounds 1 and 2 for 24 h. Values represent means S.E. These histograms are
representative of two independent experiments each performed in triplicate.
⁎
Pb0.05, significantly different from the untreated control.
positive control, HL-60 cells were also included in the experiment
and, as expected, there was an important reduction in the
proliferation of these cells (Fig. 8).
4. Discussion
Flavonoids are naturally occurring phenylbenzo-γ-pyrones
found in abundance in diets rich in fruits, vegetables and plant-
derived beverages (Middleton et al., 2000). These polyphenolic
compounds are of great current interest due to their possible
anticancer activities (Middleton et al., 2000).
In this study, we have tested five human tumor cell lines for their
sensitivity to twenty two different natural and semisynthetic
phenylbenzo-γ-pyrones derivatives. Our data indicate that betu-
letol 3-methyl ether 1 and its diacetyl derivative 2 are potent
inhibitors of proliferation and display high cytotoxic activities on
human myeloid leukemia HL-60 cells and other cell lines, such as
A431 human epidermoid carcinoma and HOS osteosarcoma cells.
Previous studies have shown that human ovarian adenocarcinoma
SK-OV-3 cells are resistant to TRAIL (Tumor Necrosis factor-
related Apoptosis-Inducing Ligand). The low sensitivity of SK-
OV-3 cells to the tested compounds could be explained by the high
levels of HER-2/neu protein and high levels of Akt (an anti-
apoptotic serine/threonine kinase that mediates survival signals)
activity in this cell line (Kim and Lee, 2005). However, the human
myeloid leukemia HL-60 cell line was especially sensitive to both
betuletol derivatives (betuletol 3-methyl ether 1 and its diacetyl
derivative 2). The introduction of a methyl group on position 4′ of
the B ring (2-phenyl group) likely contributes to this cytotoxicity

S. Rubio et al. / European Journal of Pharmacology 548 (2006) 9–20
19
explain its ineffectiveness in HOS cells. Also, among the 22
compounds presented in Table 1, betuletol 3-methyl ether 1 and
its diacetate-derivative 2 exhibited the highest cytotoxic activities
against all human cancer cell lines evaluated. In most cell lines the
IC50 values for these betuletol derivatives were lower than 10 μM,
although in HL-60 cells these values were about 1 μM.
As for all of the agents used or developed for cancer
treatment, selectivity toward cancer cells is an important
criterion. We therefore compared the effects of compounds 1
and 2 between HL-60 cells and human peripheral blood
mononuclear cells (PBMC). Interestingly, dose-response stud-
ies revealed that quiescent PBMC and proliferating PBMC were
resistant toward both betuletol derivatives 1 and 2.
In this work, we demonstrate that the exposure of HL-60
cells to betuletol 3-methyl ether and its diacetyl derivative
exerts a strong antiproliferative effect and both compounds
displayed similar IC50 values. Among the number of pathways
that can be exploited for cancer prevention and treatment,
apoptosis is a physiological process by which cells are removed
when an agent damages their DNA, therefore compounds that
can induce apoptosis may be useful in management and therapy
of cancer. Although previous studies have documented the
induction of apoptotic cell death and DNA fragmentation by
phenylbenzo-γ-pirones derivatives in the human myeloid
leukemia HL-60 cells (Lee et al., 2002) betuletol derivatives
have yet to be assessed. Our results show that betuletol 3-methyl
ether and its diacetyl derivative induced apoptosis in the
hematopoietic cell line HL-60.
Both betuletol derivatives promote the formation of apoptotic
bodies and the internucleosomal degradation of DNA, resulting in
the formation and eventual release of oligonucleosomal DNA
fragments. The apoptosis induction was accompanied by poly
(ADP-ribose) polymerase cleavage which is considered to be one
of the hallmarks of apoptosis (Nicholson and Thornberry, 1997).
We report in the present study that z-VAD-fmk, a broad-
spectrum caspase inhibitor, completely abrogated the morpho-
logical changes associated with apoptosis, DNA fragmentation
and the cleavage of poly(ADP-ribose) polymerase, which
indicated that caspases are essential components in betuletol
3-methyl ether-induced apoptosis.
Caspase-3 is the most active effector caspase, in both the
intrinsic and extrinsic pathways where it is processed and
activated by caspase-9 and caspase-8 respectively. Caspase-3
has been found to be involved in leukemia-cell apoptosis induced
by cytotoxic agents such as 1-β-D-arabinofuranosylcytosine,
mitoxantrone, etoposide and CPT-11 (Motwani et al., 1999). A
high level of caspase-3 activation and processing was found after
treatment of HL-60 cells with betuletol derivatives 1 and 2.
Previous studies have already demonstrated that most of
cytotoxic agents induce the release of mitochondrial cyto-
chrome c (Szewczyk and Wojtczak, 2002), and this protein
triggers a caspase-dependent assembly of the apoptosome (Van
Gurp et al., 2003). Although we demonstrate that betuletol
derivatives initiated redistribution of cytochrome c into the
cytosol and procaspase-9 processing, our results indicate that
the role of caspase-9 in the betuletol induced-apoptosis overall
is, at most, minimal. Moreover, we analyzed the kinetic patterns
of cytochrome c release in time course experiments after
betuletol derivatives treatments. The cytochrome c release was
not an early event in the apoptotic cell death induced by both
betuletol derivatives (results not shown). Although the exposure
of HL-60 cells to both betuletol derivatives resulted in increased
cytosolic cytochrome c, this response was not detected until
after 12 h of treatment. Consistently with this finding, the
activation of caspase-9 (as determined by a colorimetric assay
and western blot), was not observed until the same time period.
Furthermore, there have been ample examples of cross-talk
between cell death receptor-mediated signaling through the
caspase-8 and the apoptosome cascade. Caspase-8 directly
activates downstream effector caspases and also cleaves Bid and
triggers mitochondrial damage that in turn leads to cytochrome
c release. Studies have demonstrated that the intrinsic pathway
for apoptosis initiation is controlled by members of the Bcl-2
family (Marsden and Strasser, 2003; Borner, 2003), which are
crucial regulators of apoptosis in mammalian cells. However,
we were unable to demonstrate alterations in the expression or
in the mobility of Bcl-2 on sodium dodecyl sulfate-polyacryl-
amide gel electrophoresis, a phenomenon that generally
(although not invariably) accompanies perturbations in phos-
phorylation state (Haldar et al., 1995) (data not shown).
Previous studies have shown that apoptosis caused by anti-
cancer drugs may be mediated via the CD95 system (Friesen et al.,
1996), although many drugs seem to initiate the mitochondrial
pathway directly. An ample variety of mechanisms of resistance
to apoptosis that interfere at different levels of apoptosis
signalling have been described (Igney and Krammer, 2002).
Thus a search for novel agents that target aberrant apoptosis
pathways or induce alternative death pathways may be suited to
overcome treatment resistance.
Using irreversible inhibitors for selected caspases, we
attempted to further delineate the pathways of caspase activation
and their relationship to poly(ADP-ribose) polymerase cleavage
and DNA fragmentation induced by betuletol derivatives. The
effective blockage of apoptosis induction by a cell-permeable
inhibitor of caspase-8, z-IETD-fmk, but not by a cell-permeable
inhibitor of caspase-9, z-LEHD-fmk (also confirmed by kinetic
studies of caspases activation) supports a model in which
betuletol 3-methyl ether induces apoptosis by a caspase-
8 mediated mechanism.
5. Conclusion
Our results indicate that betuletol 3-methyl ether inhibits
human HL-60 cell growth and induces apoptotic cell death
involving the extrinsic pathway and could be useful in the
development of novel anticancer agents.
Acknowledgments
This work was supported by Grants from the Ministerio de
Educación y Ciencia of Spain and FEDER (SAF2004-07928),
from Consejería de Educación, Cultura y Deportes (Canary
Islands Government) and FEDER (GRUP2004/44) and Insti-
tuto Canario de Investigación del Cáncer (08/2004). We thank J.

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S. Rubio et al. / European Journal of Pharmacology 548 (2006) 9–20
Ko, C.H., Shen, S.C., Lin, H.Y., Hou, W.C., Lee, W.R., Yang, L.L., Chen, Y.C.,
2002. Flavanones structure related inhibition on TPA-induced tumor
promotion through suppression of extracellular signal-regulated protein
kinases: involvement of prostaglandin E2 in anti-promotive process. J. Cell.
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