Cytotoxic and apoptosis-inducing effect of ent-15-oxo-kaur-16-en-19-oic acid, a derivative of grandiflorolic acid from Espeletia schultzii

Article abstract of DOI:10.1016/j.phytochem.2007.07.025

ent-Kaurenic acid and many natural derivatives of this diterpene are known to have interesting biological properties. ent-15-Oxo-kaur-16-en-19-oic acid can be easily obtained from grandiflorolic acid which was first isolated from Espeletia grandiflora. The present work describes the proapoptotic effect of ent-15-oxo-kaur-16-en-19-oic acid on the human prostate carcinoma epithelial cell line PC-3 as evidenced by the changes in the expression level of proteins associated with the execution and regulation of apoptosis. Cell viability was affected upon exposure to the compound, the IC₅₀ were determined as 3.7 μg/ml, which is 4 times lower than that corresponding to a primary cell culture of fibroblasts (14.8 μg/mL). Through Western blot analysis, active forms of caspace-3 associated with the specific proteolysis of Poly(ADP-ribose) polymerase (PARP) were detected. Reduced levels of the antiapoptotic protein Bcl-2, as well as the appearance of internucleosomal DNA fragmentation, were also demonstrated. Thus, ent-15-oxo-kaur-16-en-19-oic acid may be a promising lead compound for new chemopreventive strategies, alone or in combination with traditional chemotherapy agents to overcome drug resistance in tumoral cells.

Full text of DOI:10.1016/j.phytochem.2007.07.025

Available online at www.sciencedirect.com
PHYTOCHEMISTRY
Phytochemistry 69 (2008) 432–438
www.elsevier.com/locate/phytochem
Cytotoxic and apoptosis-inducing effect of
ent-15-oxo-kaur-16-en-19-oic acid, a derivative of grandiflorolic
acid from Espeletia schultzii
a
b
c
d
Yarimar Ruiz , Juan Rodr ´ı gues , Francisco Arvelo , Alfredo Usubillaga ,
d
a
a,
*
Mariugenia Monsalve , Nardy Diez , Iv a´ n Galindo-Castro
a
Laboratorio de Gen o´ mica y Prote o´ mica, Centro de Biotecnolog ı´ a, Fundaci o´ n IDEA, Carretera Nacional Hoyo de la Puerta-Baruta, Valle de Sartenejas,
Municipio Baruta, Edo. Miranda, Caracas 1015-A, Venezuela
b
Facultad de Farmacia, UCV, Venezuela
Laboratorio de Cultivo de Tejidos y Biolog ı´ a de Tumores, Instituto de Biolog ı´ a Experimental, UCV, Venezuela
c
d
Instituto de Investigaciones, Facultad de Farmacia y Bioan a´ lisis, ULA, Venezuela
Received 10 May 2007; received in revised form 6 July 2007
Available online 14 September 2007
Abstract
ent-Kaurenic acid and many natural derivatives of this diterpene are known to have interesting biological properties. ent-15-Oxo-
kaur-16-en-19-oic acid can be easily obtained from grandiflorolic acid which was first isolated from Espeletia grandiflora. The present
work describes the proapoptotic effect of ent-15-oxo-kaur-16-en-19-oic acid on the human prostate carcinoma epithelial cell line PC-3
as evidenced by the changes in the expression level of proteins associated with the execution and regulation of apoptosis. Cell viability
was affected upon exposure to the compound, the IC50 were determined as 3.7 lg/ml, which is 4 times lower than that corresponding to a
primary cell culture of fibroblasts (14.8 lg/mL). Through Western blot analysis, active forms of caspace-3 associated with the specific
proteolysis of Poly(ADP-ribose) polymerase (PARP) were detected. Reduced levels of the antiapoptotic protein Bcl-2, as well as the
appearance of internucleosomal DNA fragmentation, were also demonstrated. Thus, ent-15-oxo-kaur-16-en-19-oic acid may be a prom-
ising lead compound for new chemopreventive strategies, alone or in combination with traditional chemotherapy agents to overcome
drug resistance in tumoral cells.
ꢀ
2007 Elsevier Ltd. All rights reserved.
Keywords: Espeletia schultzii; Espeletiinae; Frailej o´ n; ent-Kaurenoids; ent-5-Oxo-kaur-16-en-19-oic acid; Apoptosis; PC-3 cell line
1
. Introduction
lated by Piozzi et al. (1968) from Espeletia grandiflora, a
Colombian species growing around Bogota city. E. grandi-
flora is one of around 180 species of Espeletiinae (Astera-
cea), resinous plants, popularly known as frailej o´ n, that
are characteristic of the high Andean plateaus of Venezu-
ela, Colombia, and Ecuador (Cuatrecasas, 1976). In the
present work grandiflorolic acid (1b) was obtained by mild
hydrolysis of ent-15a-acetoxy-kaur-16-en-19-oic acid (1a),
isolated from Espeletia schultzii Wedd, a Venezuelan spe-
cies (Aristeguieta, 1964). This plant was first studied by
Brieskorn and P o¨ hlmann (1968) who found that its resin
contained large amounts of grandiflorolic acid (1b), and
ent-15a-acetoxy-kaur-16-en-19-oic acid (1a) was the second
Of the 20,000 plant species described in Venezuela, 1,500
of them are used for medicinal purposes (Taylor et al.,
006). ent-Kaurenic acid and many natural derivatives of
2
this diterpene are known to have interesting biological
properties (Ghisalberti, 1997). Additionally ent-15-oxo-
kaur-16-en-19-oic acid (1c) (EOKA) can be readily
obtained from grandiflorolic acid (1b) which was first iso-
*
Corresponding author. Tel.: +58 212 9035087; fax: +58 212 9035086.
E-mail address: igalindo@idea.gob.ve (I. Galindo-Castro).
0
031-9422/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.phytochem.2007.07.025

Y. Ruiz et al. / Phytochemistry 69 (2008) 432–438
433
most abundant compound. Several Espeletiinae have been
found to contain ent-15a-acetoxy-kaur-16-en-19-oic acid
1a) and grandiflorolic acid (1b) (Usubillaga et al., 2003),
but E. schultzii was chosen because it is easily collected.
Asian species pertaining to the genus Isodon (Labiatae)
have been found to contain an a,b-unsaturated ketone moi-
ety at the D ring of the kaurene skeleton (Xiang et al.,
1
1
13
14
15
1
6
17
2
0
(
1
10
8
H
2
004; Li et al., 2006). These compounds are poly-hydroxyl-
3
5
ated and most of them have been found to be cytotoxic
against several cancer cell lines. Nagashima et al. (2003)
have studied the biological properties of ent-11a-hydroxy-
kaur-16-en-15-one in a human leukemia cell line and found
evidence that this compound was able to induce apoptosis.
More recently, Rundle et al. (2006) described the ability of
EOKA (1c) to irreversibly prolong the mitotic arrest on
human epithelial tumoral cell lines, a characteristic effect
that sometimes precedes apoptosis (Sasaki et al., 2002;
Ling et al., 2002).
6
R
H
1
8
19
COOH
1
1
1
a R =
b R =
β
β
H,
H,
α
α
-OCOCH3
-OH
c R = O
Fig. 1. Chemical structures of the ent-15a-acetoxy-kaur-16-en-19-oic acid
1a), ent-15a-hydroxy-kaur-16-en-19-oic acid (grandiflorolic acid, 1b), and
ent-15-oxo-kaur-16-en-19-oic acid (1c).
(
Although many of this type of compounds have been
reported to have the capacity to induce apoptosis in differ-
ent cell lines, their molecular targets differed significantly
(
2
(
Morales et al., 2005; Castrillo et al., 2001; Lee et al.,
002), and showed dissimilar levels of drug sensitivity
Kaur et al., 2005).
1
1
20
00
Prostate cancer is recognized as one of the five move fre-
quent forms of neoplasia affecting the male population
worldwide (Jemal et al., 2005). With adenocarcinoma pro-
gression towards androgen-independence, like the PC3 cell
line, tumors become resistant to a variety of conventional
treatments (Tilley et al., 1990), demanding new therapeutic
alternatives including activation of apoptosis or pro-
grammed cell death (Honda et al., 2002; Nguyen and
Wells, 2003). The identification of potential targets that
are activated during apoptosis onset is made possible by
virtue of the selectivity of natural compounds and their
derivatives to these targets (Morales et al., 2005; Castrillo
et al., 2001; Lee et al., 2002). Considering the physiological
function that ent-kauranes may have as precursors of plant
cell proliferation regulators (Vieira et al., 2005), it is inter-
esting that they may affect animal cell proliferation as well.
In the present work, we provide evidence of the proa-
poptotic effect of EOKA (1c) on the human prostate carci-
noma epithelial cell line PC-3, which induces a differential
expression of proteins associated with the execution and
regulation of apoptosis.
8
6
4
2
0
0
0
0
0
0
5
10
15
20
25
30
ent-15-oxo-kaur-16-en-19-oic acid (μg/mL)
3
e+6
2e+6
2
1
5
e+6
e+6
e+5
0
2
. Results and discussion
0
20
40
60
Time (h)
80
100
120
We tested EOKA (1c) for its cytotoxicity against human
prostate carcinoma epithelial cell line PC-3. As can be seen
in Fig. 2a, PC-3 cells exposed to the kaurenoic acid showed
a lower viable cell number in a dose-dependent manner,
reaching an IC50 value of 3.7 lg/ml. This concentration
value is 4 times lower than that corresponding to a primary
cell culture of fibroblasts (14.8 lg/ml) (data not shown),
cells that we used to evaluate the differential effect of the
Fig. 2. Cytotoxic activity of ent-15-oxo-kaur-16-en-19-oic acid (1c) on
human prostate PC-3 cell line. (a) Dose response curve for cells treated
with EOKA (1c). The estimated IC50 value for this compound was
3
.75 lg/mL. (b) Inhibition of cell growth after treatment with EOKA (1c)
at IC50 concentration (open circles). Close circles, cell growth curve
without EOKA (1c) treatment. ANOVA:
p = 0.00001.
F (5, N = 5) =996.6,

434
Y. Ruiz et al. / Phytochemistry 69 (2008) 432–438
isolated natural compound between tumoral and normal
cells.
ments, is typical of nuclesomal DNA fragmentation pro-
duced by apoptosis.
Fig. 2b shows how EOKA (1c) exerts an antiproliferea-
tive effect on PC-3 cells when compared with untreated
cells. Such an event is more evident after 48 h of drug
exposure.
The preferential cytotoxicity of EOKA (1c) on prostate
cancerous cells over normal fibroblasts indicates a higher
sensitivity of the tumoral cells to this compound. This find-
ing is in agreement with recently reported activities for
other kauranoids such as ent-16b-17a-dihidroxykaurene
After this finding, we continued to determine whether
caspase-3, the key effector caspace of apoptosis, was also
being activated. When caspace-3 activity was specifically
determined in cells treated with EOKA (1c), this compound
was able to significantly increase (1.8 times) the specific
activity of this protease compared to the untreated cells
(see Fig. 4a).
Although basal levels of procaspase-3 have been
reported for PC-3 cell line by Winter et al. (2001), in our
study the activated forms of the cleaved polypeptide were
only evident after exposing the cells to the kaurenoic acid.
As can be seen in Fig. 4b, the active forms of the caspase-3
revealed by Western blot and represented by cleaved-poly-
peptide fragments of 17 kDa and 11 kDa are particularly
evident after the 72-h treatment, which coincides with the
activation time course of the caspace-3 enzymatic activity.
After this time, caspase-3 activity seems to decline, proba-
bly due to a generalized proteolysis caused by the caspace
cascade itself.
(
Morales et al., 2005), ent-15-oxo-1,7,14,20-tetrahydroxy-
kaur-16-ene or Kamebakaurin (Lee et al., 2002), and
ent-16-kaur-16-en-19-oic acid (Castrillo et al., 2001), deriv-
atives from kaura-9(11),16-dien-19-oic acid (Alonso, 2006),
among many others, where they are sensitizing the tumoral
cells to undergo apoptosis. Rundle et al. (2006) recently
reported that EOKA (1c) induces mitotic arrest on human
epithelial cell lines at early drug exposure times. This group
did not find evidence of apoptosis induction; however,
other compounds such as mebendazole (methyl 5-ben-
zoyl-2-benzimidazole-carbamate) (Sasaki et al., 2002), or
arsenic trioxide (Ling et al., 2002), besides inducing mitotic
arrest, also provoked apoptosis in human tumor cell lines.
To determine the possible involvement of EOKA (1c) in
apoptosis induction, we evaluated the generation of the
typical DNA ladder pattern of internucleosomal fragmen-
tation when cells undergo apoptosis. In this regard, DNA
samples from PC-3 treated cells were run by conventional
agarose gel (2.5%) electrophoresis to observe the nucleo-
somal fragmentation caused by the activation of apopto-
sis-dependent nucleases. Fig. 3 shows that cells treated
with EOKA (1c) for at least 144 h manifested a DNA frag-
mentation pattern which is not observed in control cells.
This electrophoretical pattern, as a ladder of 180-bp frag-
Poly(ADP-ribose) polymerase (PARP) is commonly
used as a native substrate of caspace-3 to follow the apop-
tosis onset. As depicted in Fig. 5, after the immunodetec-
tion of PARP by Western blotting, we observed that
PC-3 cells treated with EOKA (1c) produced the specific
cleavage of PARP, generating a fragment of 85 kDa,
0.30
0.25
0.20
0
0
0
0
.15
.10
.05
.00
0
24
48
72
96
Time (h)
1
2
3
4
5
3
3
2 kDa
7 kDa
Procaspase-3
p17
p11
GAPDH
Fig. 4. Caspase 3 activation. (a) Activity levels of caspase 3 detected in
PC-3 cells assayed with EOKA. ANOVA: H (4,N = 15) = 9.3. p = 0.0540.
(
b) Western blot of caspase-3 and procaspase-3 on PC3 cells treated with
Fig. 3. Induction of DNA fragmentation by EOKA (1c) on PC-3 cells.
DNA fragmentation was assessed on cells treated or untreated with this
compound at IC50; DNA bands were resolved by agarose gel (2.5%)
electrophoresis. (1) Molecular weight standards, (2) untreated cells (144-h
treatment with vehicle only), (3) 120-h treatment, (4) 144-h treatment.
ent-15-oxo-kaur-16-en-19-oic acid (1c). (1) Untreated cells (96-h treatment
with vehicle only), (2) 24-h treatment, (3) 48-h treatment, (4) 72-h
treatment, (5) 96-h treatment. The immunoblot of GAPDH is shown as
protein loading reference for each treatment. AMC = 7-amino-4-methyl
coumarin.

Y. Ruiz et al. / Phytochemistry 69 (2008) 432–438
435
protein Bcl-2 and the prosurvival protein Bak could
explain the commitment of the tumoral cell to undergo
apoptosis under the effect provoked by EOKA (1c). ent-
Kaurene diterpenoids isolated from Jungermannia truncata
(Nagashima et al., 2003) have the same enone moiety as
EOKA (1c) combined with hydroxyl groups at C-3, C-7,
C-11, C-14, or C-20. Since each one of these compounds
can similarly induce apoptosis, it could be inferred that
the enone moiety might be responsible for the biological
activity. Moreover, although EOKA (1c) has been proven
to cause mitotic arrest by inhibiting RanBP2 function at
early drug exposure times (Rundle et al., 2006), the specific
mechanisms of action of this compound to unchain apop-
tosis still remain to be elucidated.
Fig. 5. Western blot of PARP from PC-3 cells treated with ent-15-oxo-
kaur-16-en-19-oic acid (1c). (1) Untreated cells (96-h treatment with
vehicle only), (2) 24-h treatment. (3) 48-h treatment, (4) 72-h treatment, (5)
96-h treatment. The immunoblot of GAPDH is shown as protein loading
reference for each treatment.
containing the C-terminal end of the protein recognized by
the antibody. Control cells only showed the native unc-
leaved PARP (116 kDa). The polypeptide bands corre-
sponding to fragments of cleaved PARP became evident
after the 48-h treatment, also coinciding with the activation
time course of the caspace-3 enzymatic activity.
3. Concluding remarks
In this work we reported the differential cytotoxic and
proapoptotic effect of EOKA (1c), an ent-kaurenoic acid
obtained from a natural kaurene diterpene isolated from
an endemic plant of the Venezuelan Andes. Our study pro-
vides evidence for the role of EOKA (1c) as a potential
antitumoral agent by its ability to activate apoptosis in
PC-3 cells. We found that EOKA (1c) promotes activation
of caspace-3 and caspace specific cleavage of PARP, and
also induces nucleosomal DNA fragmentation as well as
reduction of the protein levels of Bcl-2.
Although chemotherapeutic agents have been used to
specifically kill tumor cells by inducing apoptosis, many
of these tumors acquired drug resistance to the apoptotic
stimuli during tumorigenesis. Therefore, an effective strat-
egy for overcoming the resistance to anticancer agents is
an important field of study still to be exploited. Experi-
ments are being conducted to establish the sensitizing abil-
ity of EOKA (1c) when combined with traditional drugs
for cancer treatment.
The antiapoptotic protein Bcl-2 has been proposed as
one of the key players in apoptosis regulation, particularly
in the intrinsic or mitochondrial pathway, where its higher
protein levels compared to those of proapoptotic proteins
prevent the cell to undergo apoptosis (Cory and Adams,
2
002). As shown in Fig. 6a, cells exposed to the kaurenoic
acid showed lower protein levels of Bcl-2 when compared
with untreated cells. This protein levels diminished with
the time course of the treatment. Tumor progression has
been associated with an over-expression of Bcl-2, and this
is generally linked to cells that are resistant to chemother-
apy (Johnstone et al., 2002). Similarly, in our case, EOKA
(
1c) produced a clear reduction of Bcl-2 protein levels when
compared to the untreated PC-3 cells. However, the levels
of the proapoptotic protein Bak, a Bcl-2 antagonist,
remained unchanged upon treatment with EOKA (1c)
(
see Fig. 6b). This imbalance between the antiapoptotic
4. Experimental part
4.1. Plant material
Resinous exudates (100 g) caused by wounds of moth
caterpillars on E. Schultzii Wedd were collected from
plants in Mucubaj ´ı , Merida State, at 3500 m above sea
level in March 2004. A voucher specimen of the plant is
kept at the MERF Herbarium (No. 1038).
4.2. Instrumentation
Melting points were measured on a Fisher Johns hot
stage and are uncorrected. Optical rotations were taken
on a Jasko electropolarimeter Model DIP 370. IR spectra
were measured on a Perkin Elmer 1720X apparatus as
KBr discs. H and C NMR measurements were per-
formed on a Bruker Advance DRX-400. UV spectra were
Fig. 6. Western blot of Bcl-2 and Bak proteins on PC-3 cells treated with
ent-15-oxo-kaur-16-en-19-oic acid (1c). (1) Untreated cells (96-h treatment
with vehicle only), (2) 24-h treatment, (3) 48-h treatment, (4) 72-h
treatment, (5) 96-h treatment. The immunoblot of GAPDH is shown as
protein loading reference for each treatment.
1
13

436
Y. Ruiz et al. / Phytochemistry 69 (2008) 432–438
measured on a Shimadzu UV-1700 spectrometer. EIMS
were obtained on a Hewlett–Packard model 5973 spec-
trometer at 70 eV. Flash chromatography was performed
on silica gel Merck 60 (230–400 mesh), TLC was carried
out on silica gel Merck 60 F254.
m, H-2b), 1.38 (1H, m, H-14b), 1.32 (1H, m, H-7b), 1.22
(3H, s, H-18), 1.17 (1H, m, H-9), 1.0 (1H, m, H-3b), 0.99
1
3
(3H, s, H-20), 0.78 (1H, dt, J = 4, 14 Hz, H-1a),
C
NMR (CDCl , ppm): 211.0 (C-15), 184.4 (C-19), 149.5
3
(C-16), 114.5 (C-17), 56.0 (C-9), 52.5 (C-8), 51.6 (C-5),
43.7 (C-4), 40.3 (C-10), 39.8 (C-1), 38.1 (C-13), 37.6 (C-
4
.2.1. ent-15-Oxo-kaur-16-en-19-oic acid (EOKA) (1c)
Resin (100 g) from E. schultzii was extracted twice at
3), 36.5 (C-14), 32.6 (C-7), 32.2 (C-12), 28.9 (C18), 20.0
(C-6), 18.8 (C-2), 18.4 (C-11), 15.5 (C-20) ; MS (m/z, %):
+
room temperature with n-hexane (0.5 l). The solvent was
316 (M , C H O , 100), 301 (28), 283 (16), 255 (26),
2
0
28
3
vacuum distilled to yield 58 g of extract. This extract was
207 (17), 148 (52). 121 (30).
dissolved Et O (1 l) and extracted with 0.5 N aqueous
2
NaOH (2 · 0.5 l). The aqueous phase was neutralized by
addition of diluted HCl (1 l) to pH 3.0. Upon cooling,
the acid fraction was recovered by extracting shaking twice
4.3. Culture cell lines
with Et O. The combined ether extract were dried
Androgen-independent human prostate carcinoma epi-
thelial cell line PC-3 used in this study was kindly provided
by Dr. Marie France Poupon at Institute Curie, Paris-
France, and was maintained in RPMI (Roswell Park
Memorial Institute) medium supplemented with 10% fetal
bovine serum, 1% of L-glutamin and 1% streptomycin (all
obtained from Sigma–Aldrich, USA). Cells were grown
2
(
Na SO ) and distilled to yield 28 g of a yellow mixture.
2 4
The acid fraction (20 g) was submitted to flash cc on silica
gel (500 g). Elution started with n-hexane:diethylether (3:1,
v/v) and 200 ml fractions were taken and inspected by
TLC. Fractions 45–87 yielded 2.7 g of pure ent-15a-acet-
oxy-kaur-16-en-19-oic acid (Fig. 1a), mp 172–174 ꢁC
ꢀ
1
(
1
recrystallized from EtOH), IR (KBr) mmax cm : 1725,
in a humidified incubator with 5% CO and 95% air at
2
1
690, 1650, 690; and H-NMR (CDCl , 400 MHz) d 5.27
37 ꢁC.
3
(
1H, s, H-17a), 5.10 (2H, s, H-17b and H-15), 2.81 (1H,
br s, H-13), 2.08 (3H, s, COCH ), 1.25 (3H, s, H-18),
3
1
0
.95 (3H, s, H-20). Melting point, IR and H NMR signals
4.4. Cytotoxicity assay
agree with those reported by Brieskorn and P o¨ hlmann
(
(
1968). ent-15a-Acetoxy-kaur-16-en-19-oic acid (1a)
0.35 g) was dissolved in 25 ml of 0.5 N KOH in MeOH
A 96-well microtiter plate (tissue culture grade) contain-
ing 0.2 ml of growth medium/per well (RPMI) was seeded
with sufficient PC-3 cells to provide approximately 70%
growth confluence after 24–48 h of culture. At this point,
cells were exposed to EOKA for 72 h at concentrations
ranging from 5 to 25 lg/ml, and then evaluated for cyto-
toxicity. In all cases, although EOKA (1c) was dissolved
in dimethylsulfoxide (DMSO), the final concentration of
this compound in the culture medium was lower than
1%, a concentration that has neither cytotoxic effect nor
causes any interference with the colorimetric detection
method.
and left overnight at room temperature. The solvent was
distilled; the residue was treated with diluted HCl (25 ml);
and then extracted with Et O (2 · 50 ml). Distillation of
the Et o extract yielded ent-15a-hydroxy-kaur-16-en-19-
2
2
oic acid (308 mg) (Fig. 1b) which was crystallized from
ꢀ
1
MeOH, mp 224–228 ꢁC, IR (KBr) mmax cm : 3350, 1695,
96. The IR spectrum was identical to that reported by Pio-
8
zzi et al. (1968) for grandiflorolic acid (1b).
A solution containing 200 mg (0.63 mmol) of ent-15a-
hydroxy-kaur-16-en-19-oic acid (1b) in pyridine (5 ml)
was treated with CrO (250 mg, 2.5 mmol) in pyridine
Cytotoxicity assays were carry out by colorimetry fol-
lowing the reduction of 3-(4,5-dimethylthiazol-2-yI)-2,5-
diphenyltetrazolium bromide (MTT assay) (Denizot and
Lang, 1986). This colorimetric assay is based on the ability
of live, but not dead, tumor cells to reduce dissolved MTT
into an insoluble purple compound, formazan, by cleavage
of the tetrazolium ring by dehydrogenase enzymes. Cells
grown in microtiter plates were first incubated with MTT
at 37 ꢁC for 3 h, then they were washed with phosphate
buffered saline (PBS), and finally the colorimetric detection
was done by the addition of Formazan (dissolved in 1%
dimethyl sulfoxide, DMSO). Absorbance was measured
3
0
(
Sarett s reagent) and left overnight at room temperature.
The following day, in order to end the reaction, H O was
2
added to the reaction mixture (25 ml) which was filtered
and extracted with Et O (3 · 50 ml). The condensed ether
2
extracted were dried over (anhyd Na SO ) and evaporated
2
4
to dryness. The crude product was submitted to flash
chromatography, which was eluted with 1-hexane:ether
(
3:1, v/v) to yield ent-15-oxo-kaur-16-en-19-oic acid (190
25
mg) (Fig. 1c), mp 184–185 ꢁC, ½aꢁ -165 (c 0.15, CHCl ),
D
3
UV k
234 nm (loge 3.85) typical of an enone system
max
ꢀ
1
on a five member ring, IR (KBr) mmax cm : 2940, 2866,
1
1
ꢂ
723, 1690, 1645, 1466, 1256, 939, H NMR (CD Cl,
by a microplate reader (SepctraFluor , Tecan) set at a
3
4
00 MHz): 5.91(1H, s, H-17a), 5.22 (1H, s, H-17b), 3.02
wavelength of 570 nm. The IC50 value in the MTT assay
was defined as the concentration of test compound result-
ing in a 50% reduction of absorbance compared to
untreated cells. Further evaluations of the EOKA on the
(
(
1H, br s, H-13), 2.37 (1H, d, J = 11; 9 Hz, H-14a), 2.12
1H, m, H-3a), 1.84 (1H, m, H-1b), 1.80 (1H, m, H-6b),
1
1
.78 (1H, m, H-7a), 1.65 1H, m, H-11b), 1.63 (1H, m, H-
2b), 1.45(1H, m, H-2a), 1.44 (1H, m, H-6a), 1.40 (1H,
cell line were performed using the IC50
.

Y. Ruiz et al. / Phytochemistry 69 (2008) 432–438
437
4
.5. Effect of EOKA (1c) on cell growth
4.9. Caspace-3 enzymatic activity determination
6
6
Cells (10 ) were plated in 35-mm tissue culture dishes at
Cultured cells (10 ) were washed with PBS at 37 ꢁC and
resuspended in PBS at 4 ꢁC. Cells were harvested by centri-
fugation at 4 ꢁC (450·g, 10 min) and tested with the fluoro-
metric CasPACE Assay System (Promega). Briefly, the cell
pellet was resuspended in hypotonic cell lysis buffer at a
3
7 ꢁC in Dulbecco’s Modified Eagle’s Medium (DMEM)
containing EOKA at 3.7 lg/ml (IC ). The number of via-
5
0
ble cells per dish was counted with a hemocytometer at 24-
h intervals for a period of 96 h. Cells were collected from
culture dishes after a trypsin-EDTA treatment for 10 min
at 37 ꢁC. All experiments were done in duplicate and each
set was repeated at least twice.
8
final concentration of 10 cells/ml and centrifuged
(16,000·g, 4 ꢁC, 20 min). The supernatant was preincu-
bated for 30 min at 30 ꢁC with the caspase assay buffer.
The samples were then incubated with the caspase-3 sub-
strate Ac-DEVD-AMC for 1 h at 30 ꢁC. Fluorescence
intensity was measured with the aid of a microplate reader.
The specificity of the assay was verified by adding the cas-
pase-3 inhibitor Ac-DEVD-CHO to the incubation mix-
ture. Caspase-3 specific activity was measured as the
amount of the free fluorochrome 7-amino-4-methyl couma-
4
.6. Statistical analysis
Comparisons between the means of various treatment
groups were analyzed by one-way ANOVA. The difference
among means was considered significant when p < 0.05.
ꢀ
1
ꢀ1
4
.7. Nucleosomal DNA fragmentation assay
rin (AMC) released · min · lg of protein.
PC-3 cells were grown until 70% confluent and were
then treated with EOKA (1c) or vehicle (1% DMSO) as
negative control. Cells were collected by centrifugation
Acknowledgements
(
1500g · 20 min), washed with cold phosphate-buffered sal-
The authors thank Sharon Sumpton for revising the
English text and Professor Jos e´ Luis Ramirez for helpful
suggestions on the manuscript.
ine and lysed in Lysis Buffer (50 mM Tris, pH 7.4, 5 mM
EDTA and 1% SDS) for 20 min on ice. The supernatant
was treated with 50 lg RNase A at 37 ꢁC for 30 min and
then with Proteinase K at 0.1 mg/ml for another hour.
After phenol/CHCl (1:1, v/v) extraction, DNA was pre-
cipitated with i-PrOH. The DNA pellet was rinsed with
3
References
EtOH–H O (7:3, v/v) air dried, and dissolved in TE buffer
2
Alonso, R., 2006. Synthetic diterpene derivatives from kaura-9(11),16-
dien-19-oic acid: cytostatic and cytotoxic activity against human
cancer cell lines. Khim.-Farm. Zh. 40, 14–17.
Aristeguieta, L., 1964. Flora de venezuela, edicion especial del instituto
bot a´ nico de venezuela. Compositae X, 447–448.
Brieskorn, C., P o¨ hlmann, E., 1968. Diterpene vom kaurantyp aus der
composite Espeletia schultzii (wedd). Tetrahedron Lett. 54, 5661–5664.
Castrillo, A., De las Heras, B., Hortelano, S., Rodr ´ı guez, B., Villars, A.,
Bosc a´ , L., 2001. Inhibition of the nuclear factor jB (NFjB) pathway
by tetracyclic kaurene diterpene in macrophages. J. Biol. Chem. 276,
(
10 mM Tris–HCl, 1 mM EDTA, pH 7.3). The DNA frag-
ments (10 lg) were separated by conventional electropho-
resis on a 2.5% agarose gel and visualized after
ꢂ
SYBRGreen staining (Sigma–Aldrich).
4
.8. SDS-PAGE and western blot analysis
6
PC-3 cells (2 · 10 ) treated and untreated with ent-OKA
1c) were lysed by adding Lysis Buffer RIPA (150 mM
1
5854–15860.
(
Cory, S., Adams, J., 2002. The Bcl2 family: regulators of the cellular life-
or-death switch. Nature Rev. Cancer 2, 647–656.
Cuatrecasas, J., 1976. A new subtribe in the Heliantheae (Compositae)
Espeletiinae. Phytologia 35, 43–61.
NaCl; 50 mM Tris pH 8; 0,1% SDS; 0.5% sodium deoxico-
late; 1% Nonidet P40), centrifuged to separate non-soluble
material, and then equal amounts of soluble proteins were
subjected to SDS-polyacrylamide gel electrophoresis (SDS-
PAGE) and then transferred onto a Hybond-P membrane
Denizot, F., Lang, R., 1986. Rapid colorimetric assay for cell grow and
survival modifications to the tetrazolium dye procedure giving
improved sensitivity and reliability. J. Immunol. Meth. 89, 271–277.
Ghisalberti, E.L., 1997. The biological activity of naturally occurring
kaurene diterpenes. Fitoterapia LXVIII, 303–325.
Honda, T., Shunsuke, K., Spurgers, K., Gjertsen, B., Roth, J., Fang, B.,
Lowe, S., Norris, J., Meyn, R., McDonnell, T., 2002. A recombinant
adenovirus expressing wild-type Bax induces apoptosis in prostate
cancer cells independently of their Bcl-2 status and androgen sensitiv-
ity. Cancer Biol. Ther. 1, 163–167.
(
Amersham Pharmacia) with the use of a Mini Trans-Blot
ꢂ
Cell (Bio-Rad). The membranes were blocked with TBST
50 mM Tris–HCl, pH 7.5, 150 mM NaCl, and 0.001%
(
Tween 20) containing 5% (w/v) nonfat milk, washed, and
probed with the appropriate concentration of primary anti-
bodies (1:400 dilution, Santa Cruz Biotechnology) for 2 h
at 4 ꢁC. After washing with TBST, the membrane was incu-
bated with 1:2000 diluted peroxidase-conjugated goat anti-
Jemal, A., Murray, T., Ward, E., Samuels, A., Tiwari, R., Ghafoor, A.,
Feuer, E., Thun, M., 2005. Cancer statistics, 2005. Cancer J. Clin. 55,
1
0–30.
(
mouse IgG) Ig or goat anti-(rabbit IgG) Ig (Santa Cruz
Johnstone, R., Ruefli, A., Lowe, S., 2002. Apoptosis: a link between
cancer genetics and chemotherapy. Cell 108, 153–164.
Kaur, S., Hushem, M., Arora, S., H a¨ rk o¨ nen, P., Kumar, S., 2005. The
in vitro cytotoxic and apoptotic activity of Triphala, an Indian herbal
drug. J. Ethnopharmacol. 97, 15–20.
Biotechnology) for 1 h at 25 ꢁC. The antibody reactive
bands were revealed using the SuperSignal West Pico
Chemiluminescent Substrate (Pierce) according to the man-
ufacturer’s instructions.

438
Y. Ruiz et al. / Phytochemistry 69 (2008) 432–438
Lee, J., Koo, T., Hwang, B., Lee, L., 2002. Kaurene diterpene,
Kamebakaurin, inhibits NF-(B by directly targeting the DNA-binding
activity of p50 and blocks the expression of antiapoptotic NF-jB
target genes. J. Biol. Chem. 277, 18411–18420.
Li, X., Xiao, W., Pu, J., Ban, L., Shen, Y., Weng, Z., Li, S., Sun, H., 2006.
Cytotoxic ent-kaurene diterpenoids from Isodon phyllostachys. Phyto-
chemistry 67, 1336–1340.
that inhibits mitotic chromososme movement and binds the
kinetochore protein Ran-binding protein 2. ASC Chem. Biol. 7,
443–450.
Sasaki, J., Ramesh, R., Chada, S., Gomyo, Y., Roth, J., Mukhopadhyay,
T., 2002. The anthelmintic drug mebendazole induces mitotic arrest
and apoptosis by depolymerizing tubulin in non-small cell lung cancer
cells. Mol. Cancer Ther. 1, 1201–1209.
Ling, Y.-H., Jiang, J.-D., Holland, J., Perez-Soler, R., 2002. Arsenic trioxide
produces polymerization of microtubules and mitotic arrest before
apoptosis in human tumor cell lines. Mol. Pharmacol. 62, 529–538.
Morales, A., P e´ rez, P., Mendoza, R., Compagnone, R., Su a´ rez, A.,
Arvelo, F., Ramirez, J., Galindo-Castro, I., 2005. Cytotoxic and
propapoptotic activity of ent-16b-17a-dihydroxykaurane on human
mammary carcinoma cell line MCF-7. Cancer Lett. 218, 109–116.
Nagashima, F., Kondoh, M., Kawase, M., Simizu, S., Osada, H.,
Watanabe, M., Sato, M., Asakawa, Y., 2003. Apoptosis-inducing
properties of ent -kaurene-type diterpenoids from the liverwort
Jungermannia truncate. Planta Med. 69, 377–379.
Taylor, P., Cesari, I., Arsenak, M., Ballen, D., Abad, M., Fern a´ ndez, A.,
Milano, B., Ruiz, M., Williams, B., Michelangeli, F., 2006. Evaluation
of Venezuelan medicinal plant extracts for antitumor and antiprotease
activities. Pharm. Biol. 44, 1–14.
Tilley, W., Wilson, M., Marcelli, M., McPhaul, M., 1990. Androgen
receptor gene expression in human prostate carcinoma cell lines.
Cancer Res. 50, 5382–5386.
Usubillaga, A., Romero, M., Aparicio, R., 2003. Kaurenic acid in
Espeletiinae. Acta Hort. 597, 129–130.
Vieira, H., Takahashi, J., Pimenta, L., Boaventura, M., 2005. Effects of
kaurane diterpene derivatives on germination and growth of Lactuca
sativa seedlings. Z. Naturforsch. C 60, 72–78.
Winter, R., Kramer, A., Borkowski, A., Kyprianou, N., 2001. Loss of
caspase-1 and caspase-3 protein expression in human prostate cancer.
Cancer Res. 61, 1227–1232.
Xiang, W., Li, R.-T., Wang, Z.-Y., Li, S.-H., Zhao, Q.-S., Zhang, H.-J.,
Sun, H.-D., 2004. ent-kaurene diterpenoids from Isodon aresbius.
Phytochemistry 65, 1173–1177.
Nguyen, J., Wells, A., 2003. Direct activation of the apoptotic machinery
as a mechanism to target cancer cells. Proc. Natl. Acad. Sci. USA 100,
7533–7538.
Piozzi, F., Sprio, V., Passannanti, S., Mondelli, R., 1968. Structura
dell’acido gradiflorolico. Gazz. Chim. Ital. 98, 907–910.
´
Rundle, N., Nelson, J., Flory, M., Joseph, J., Thng, J., Aebersold,
R., Dasso, M., Andersen, R., Roberge, M., 2006. An ent-kaurene