Imbricatolic acid from Juniperus communis LPrevents cell cycle progression in CaLu-6 cells

Article abstract of DOI:10.1055/s-0030-1271104

Imbricatolic acid was isolated from the methanolic extract of the fresh ripe berries of Juniperus communis (Cupressaceae) together with sixteen known compounds and a new dihydrobenzofuran lignan glycoside named juniperoside A. Their structures were determined by spectroscopic methods and by comparison with the spectral data reported in literature. Imbricatolic acid was evaluated for its ability to prevent cell cycle progression in p53-null CaLu-6 cells. This compound induces the upregulation of cyclin-dependent kinase inhibitors and their accumulation in the G1 phase of the cell cycle, as well as the degradation of cyclins A, D1, and E1. Furthermore, no significant imbricatolic acid-induced apoptosis was observed. Therefore, this plant-derived compound may play a role in the control of cell cycle. Georg Thieme Verlag KG Stuttgart · New York.

Full text of DOI:10.1055/s-0030-1271104

1
822 Original Papers
Imbricatolic Acid from Juniperus communis L.
Prevents Cell Cycle Progression in CaLu-6 Cells
1
2
1
1
2
2
Authors
Simona De Marino *, Fabio Cattaneo *, Carmen Festa , Franco Zollo , Annalisa Iaccio , Rosario Ammendola ,
3
3
Filomena Incollingo , Maria Iorizzi
1
Affiliations
Dipartimento di Chimica delle Sostanze Naturali, Università degli Studi di Napoli Federico II, Napoli, Italy
Dipartimento di Biochimica e Biotecnologie Mediche, Università degli Studi di Napoli Federico II, Napoli, Italy
Dipartimento di Scienze e Tecnologie per lʼAmbiente e il Territorio, Università degli Studi del Molise, Pesche (Isernia), Italy
2
3
Key words
Abstract
cells. This compound induces the upregulation of
cyclin-dependent kinase inhibitors and their ac-
cumulation in the G1 phase of the cell cycle, as
well as the degradation of cyclins A, D1, and E1.
Furthermore, no significant imbricatolic acid-in-
duced apoptosis was observed. Therefore, this
plant-derived compound may play a role in the
control of cell cycle.
"
l Juniperus communis
!
"
l Cupressaceae
Imbricatolic acid was isolated from the metha-
nolic extract of the fresh ripe berries of Juniperus
communis (Cupressaceae) together with sixteen
known compounds and a new dihydrobenzofu-
ran lignan glycoside named juniperoside A. Their
structures were determined by spectroscopic
methods and by comparison with the spectral da-
ta reported in literature.
"
l diterpenes
"
l dihydrobenzofuran
lignan glycoside
l CDK inhibitors
"
"
l cell cycle
Supporting information available online at
http://www.thieme-connect.de/ejournals/toc/
plantamedica
Imbricatolic acid was evaluated for its ability to
prevent cell cycle progression in p53-null CaLu-6
Introduction
CDK inhibitors (CKIs) [10]. Two CKI gene families
have been defined. The INK4 gene family encodes
!
INK4a
INK4b
INK4c
INK4d
Several medicinal plants, such as Juniperus sp.
p16
, p15
, p18
, and p19
, all of
(
Cupressaceae) [1], contain metabolites able to in-
which bind to CDK4 and CDK6 and inhibit kinase
activities by interfering with their association
duce cell cycle arrest, playing an important role in
cancer prevention. Many reports are focused on
the determination of the chemical composition
of the berryʼs essential oil [2,3], and limited data
are reported on nonvolatile components. Chemi-
cal components of the methanol extract from
Juniperus communis berries include labdane
monoterpene and megastigmane glycosides [4],
labdane diterpenes [5], flavonoids, and biflavo-
noids [6]. Labdane diterpenes, isolated from sev-
eral plant families, show a variety of biological ac-
tivities [7] including the inhibition of cell prolifer-
ation in several cell lines [8]. In this study we ana-
lyzed the methanolic extract of berries from
J. communis and focused on the bioactivity of im-
bricatolic acid, a labdane diterpene isolated as a
major component.
with D-type cyclins [9]. In contrast, CKIs of the
Cip/Kip family (p21Cip1/Waf1/Sdi1, p27
Kip1
, and
Kip2
p57 ) bind to both cyclins and CDK subunits
and can modulate the activities of cyclin D-, E-,
received
revised
accepted
July 22, 2010
April 4, 2011
April 18, 2011
Cip1/Waf1/Sdi1
A-, and B-CDK complexes [9]. p21
gene
expression is regulated by transcriptional and
posttranscriptional mechanisms. In tumor cells
Bibliography
which lack p53 or with a mutant form of p53,
DOI http://dx.doi.org/
0.1055/s-0030-1271104
Cip1/Waf1/Sdi1
p21
is activated through p53-indepen-
1
Published online May 12, 2011
Planta Med 2011; 77:
dent pathways.
In the present paper, we report: i) the isolation
and structural elucidation of a new compound
and eleven known components from a metha-
nolic extract of berries from J. communis, and ii)
the ability of imbricatolic acid, a known labdane
diterpene, to induce CKIs upregulation, accumu-
lation in G1 phase of the cell cycle and cyclins
degradation in p53-null human lung tumor cells.
1
822–1828 © Georg Thieme
Verlag KG Stuttgart · New York ·
ISSN 0032‑0943
Correspondence
Prof. Maria Iorizzi
Dipartimento di Scienze
e Tecnologie per lʼAmbiente
e il Territorio (DiSTAT)
Università degli Studi del Molise
Contrada Fonte Lappone
Cyclin/cdk (cyclin dependent kinase) complexes
facilitate progression through the cell cycle and
are activated at specific checkpoints [9]. These
complexes are also regulated by their binding to
8
6090 Pesche (Isernia)
Italy
Phone: + 390874404100
Fax: + 390874404123
iorizzi@unimol.it
* These authors contributed equally to this work.
De Marino S et al. Imbricatolic Acid from… Planta Med 2011; 77: 1822–1828

Original Papers 1823
Materials and Methods
Table 1 DCCC fractionation and HPLC purification of CHCl3 and n-BuOH ex-
tracts.
!
General experimental procedures
High-resolution fast atom bombardment mass spectrometry
Fraction
Amount (mg)
CHCl3 extracta
Compound
MeOH/H2O
(HRFAB‑MS) was recorded on a Fisons VG Prospec instrument,
and electrospray ionization mass spectrometry (ESI‑MS) experi-
ments were performed on an Applied Biosystem API 2000 triple-
quadrupole mass spectrometer. Optical rotations were deter-
mined on a Jasko P-2000 polarimeter. H and C NMR spectra
were determined on a Varian Unity INOVA spectrometer at
1
2
2
3
3
4
4
4
5
5
5
1.2
17
16
8
1:1
7:3
8:2b
1.5
1.7
1
13
5.8
2
7:3
1.8
4
45:55b
8:2
3.0
10
11
12
9
500.13 and 125.77 MHz, respectively, equipped with an indirect
1.0
8:2
detection probe. Chemical shifts were referenced to the solvent
1.2
8:2
signals: deuterated methanol (CD OD). GC analyses were per-
3
1.8
75:25
75:25
75:25
formed on an Agilent Technologies 6850 Series II gas chromato-
graph for capillary column (HP-5, 30 m × 0.25 mm, 180°; helium
1.0
5
2.0
3
−1
n-BuOH extract
a
carrier flow 10 mL min ) and a FID detector operated at 260°.
Droplet countercurrent chromatography (DCCC) was performed
on a DCC‑A apparatus (Tokyo Rikakikai Co.). HPLC was per-
formed using a Waters 510 pump equipped with a Waters U6K
injector and a Waters 401 differential refractometer as the de-
tector, using a C18 µ-Bondapak column (30 cm × 3.9 mm; i.d.;
A
B
C
D
E
1.3
1.4
3.2
1.3
3.0
7
1
4:6
1:1
8:2
1:1
15
14
13
a
All fractions were purified on C18 µ-Bondapak column. b Fraction purified on a Luna
−1
flow rate 1 mL min ; Waters) and a Luna C-18 column (3 µ,
−
1
C‑18 column
1
50 × 4.60 mm i.d.; flow rate 1 mL min ; Phenomenex).
Plant material
Ripe berries of Juniperus communis L. (Cupressaceae) were col-
lected in the mountain areas of Isernia (Italy) in October 2008
and identified by Dr. Paola Fortini. A voucher specimen is depos-
ited (JC-486-08) at the Herbarium of DiSTAT, University of Molise
MeOH/H O (80:18:2). Five fractions (A–E) were obtained and
purified by HPLC as summarized in l Table 1. The purity of com-
pounds 1–17 was greater than 95% determined by HPLC method
2
"
(see experimental section), MS, and NMR. The bold number in
"
(Pesche). Berries were kept frozen at −20°C until analyzed.
brackets refers to the chemical structures indicated in l Fig. 1.
Copies of original spectra can be obtained from the author of cor-
respondence.
Extraction and isolation
Fresh ripe berries (400 g) were crushed and extracted with MeOH
(3 × 2 L) at room temperature for 24 h. The combined extracts
Acid hydrolysis of 1
(170 g) were concentrated and subjected to a modified Kupchanʼs
Compound 1 (0.5 mg) was hydrolyzed with 2N CF CO H (2 mL) at
3
2
partition methodology as described [11]. The MeOH extract was
dissolved in 10% aqueous methanol and partitioned against n-
hexane (3 × 400 mL) yielding 1.3 g of extract. The water content
110° in a sealed tube for 8 h. After cooling, the solution was di-
luted with H O (5 mL) and extracted with AcOEt (3 × 2 mL). The
2
aqueous layer was evaporated to dryness under reduced pres-
sure, and the residue was reacted with 0.1 M L-cysteine methyl
ester hydrochloride in anhydrous pyridine (200 µL) for 1 h at 60°
[12]. 1-(Trimethylsilyl)imidazole in pyridine was added, and the
thiazolidine derivatives analyzed by GC. L-rhamnose was con-
firmed in 1 by comparison of the retention time of their deriva-
(% v/v) of the MeOH extract was adjusted to 40% and partitioned
against CHCl (5 × 400 mL), yielding 1.5 g of extract; the aqueous
3
residue was concentrated and partitioned against n-BuOH
(3 × 500 mL) to give 2.0 g of n-BuOH extract. The n-hexane extract
(
1.3 g) was separated by column chromatography on SiO (50 g,
2
2
30–400 mesh silica gel; 1.5 × 45 cm) and stepwise eluted using
n-hexane/EtOAc with the ratio of 100:0 (300 mL), 99:1, 98:2,
0:20 (each 150 mL), 96:4, 95:5, 50:50, 0:100 (each 200 mL)
tives with those of D-rhamnose (t = 12.60 min) and L-rhamnose
(t = 12.14 min).
R
R
8
to give 8 corresponding fractions (A–H). 150 × 10 mL tubes were
Reagents and cell culture treatments
collected and combined on the basis of their similar TLC behavior
SDS-PAGE reagents were from Bio-Rad. Anti-active phosphoryl-
(
SiO with n-hexane/EtOAc 95:5). Fraction H (70 mg) was then
ated ERK1/2, anti-tubulin, anti-PKCδ, anti-cyclinA, anti-cyclinB,
2
Cip1/Waf1/Sdi1
Kip
purified by HPLC (C18 µ-Bondapak column; MeOH/H O 8:2 as
eluent, flow rate 1 mL/min) to give mainly cis-communic acid (6,
anti-cyclinD1, anti-cyclinE1, anti- p21
, anti-p27
,
2
INK4a
anti-p16
, anti-EGFR, anti-PARP‑1, anti-caspase3, and anti-
2
.8 mg). The CHCl extract (1.5 g) was fractionated by DCCC using
rabbit antibodies were obtained from Santa Cruz Biotechnology
Inc. Protein A-horseradish peroxidase and anti-mouse Ig-horse-
radish peroxidase were from Amersham Pharmacia. PD098059
3
CHCl /MeOH/H O (7:13:8) in the ascending mode (the lower
3
2
phase was the stationary phase); 320 tubes (6 mL) were col-
lected, flow rate 18 mL/h. Fractions were combined and moni-
tored by TLC on SiO with CHCl /MeOH/H O (80:18:2) as eluent
to give five main fractions summarized in l Table 1. The n-BuOH
extract (2 g) was submitted to DCCC with n-BuOH/Me CO/H O
®
and rottlerin were from Calbiochem. PepTag assay for nonra-
dioactive detection of protein kinase C was from Promega. Hu-
man anaplastic lung cancer cells CaLu-6 were purchased from
ATCC and grown in Dulbeccoʼs modified Eagleʼs medium (DMEM)
containing 10% fetal bovine serum (FBS), 100 U/mL penicillin,
100 µg/mL streptomycin, 1% L-glutammin, and 1% modified Ea-
gleʼs medium (MEM).
2
3
2
"
2
2
(
3:1:5) in the descending mode (the upper phase was the sta-
tionary phase). 210 tubes (6 mL) were collected, flow rate
8 mL/h. Fractions were combined and monitored by TLC on Sili-
ca gel plates with n-BuOH/OHAc/H O (12:3:5) and CHCl /
1
2
3
De Marino S et al. Imbricatolic Acid from… Planta Med 2011; 77: 1822–1828

1
824 Original Papers
Fig. 1 Natural compounds isolated from Juniperus
communis L. berries.
Imbricatolic acid was diluted in DMSO at a final concentration of
Cell viability
4
10 mM. Cells were grown until they reached 80% confluence and
Calu-6 cells were placed (4 × 10 cells per well) in 96-well plates
(Corning) and incubated at 37°C in 200 µL of complete culture
medium containing 10 µM imbricatolic acid or 0.1% DMSO for 3,
12, 24, 48, and 72 hrs. 3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyl
tetrazoliumbromide (MTT; 5 mg/mL in PBS) was added to each
well and incubated for 4 h. After removal of the medium, 0.2 mL
of DMSO was added to each well. The absorbance of the resulting
formazan salts was recorded on a microplate reader at the wave-
length of 540 nm. The effect of imbricatolic acid on growth inhi-
bition was assessed as percent cell viability where DMSO-treated
cells were taken as 100% viable. DMSO at the concentrations used
was without any effect on cell viability. Four independent experi-
ments were performed.
successively stimulated by imbricatolic acid at the final concen-
tration of 10 µM for different times, as indicated in the figures.
In other experiments, growing cells were preincubated with
50 µM PD098059 for 90 min, or with 1 µM rottlerin for 1 h, before
the stimulation with 10 µM imbricatolic acid.
Western blot analysis and protein kinase C activity assay
Total cellular lysates and cell membranes were purified from Ca-
Lu-6 cells as previously described [13]. Western blot experiments
were performed in triplicate, as previously described [13]. The
expression of targeted proteins was detected by an ECL kit (Amer-
sham Biosciences) and visualized by autoradiography. Protein ki-
nase C activity assay was performed accordingly to manufac-
turerʼs instructions (Materials and Methods 1S, Supporting Infor-
mation).
Analysis of apoptosis and cell cycle
Analysis of DNA content by propidium iodide incorporation was
performed in permeabilized cells, as described [14] (Materials
and Methods 2S, Supporting Information).
De Marino S et al. Imbricatolic Acid from… Planta Med 2011; 77: 1822–1828

Original Papers 1825
Table 2 1H and 13C NMR data (CD3OD, 500 and 125 MHz) of compound 1.
Position
δH (J in Hz)
δC
HMBC
1
2
3
4
5
6
7
–
138.8
110.9
151.7
146.1
119.2
118.7
89.0
7.06 br s
–
C4, C6, C7
–
7.08 d (8.4)
6.94 d (8.4)
5.57 d (5.7)
C1, C3
C2, C4, C7
C1, C2, C6, C8, C9,
C3′, C5′
8
9
3.47 ovl
55.6
64.8
3.77 dd (9.9, 17.9),
C7, C8, C5′
3
.88 ovl
1
2
3
4
5
6
7
8
9
′
′
′
′
′
′
′
′
′
–
136.4
116.7
146.0
140.7
129.1
116.3
32.4
6.59 s
–
C4′, C6′, C7′
–
–
6.62 s
2.58 d (7.3)
1.80 m
3.58 t (6.5)
3.83 s
C8, C2′
C1′, C2′, C6′, C8′, C9′
C1′, C7′, C9′
C7′, C8′
35.5
62.0
OCH3
56.0
C5
Rhamnose
1′′
2′′
3′′
4′′
5′′
6′′
5.35 br s
4.08 br s
3.90 ovl
101.1
71.7
71.9
73.5
70.5
17.6
C3′, C3′′, C5′′
3.47 ovl
3.82 ovl
1.22 d (6.2)
C4′′, C5′′
1H and 13C assignments achieved by COSY, TOCSY, HSQC, and HMBC experiments
Results and Discussion
Fig. 2 CaLu-6 cells were exposed to 10 µΜ imbricatolic acid (IA) for the
!
indicated times, and 40 µg of cell lysates were loaded on 12% SDS-PAGE.
A The upregulation of p21Cip1/Waf1/Sdi1, p27Kip1, and p16INK4a and B degra-
dation of cyclin A, cyclin D1, and cyclin E1 were immunodetected with
specific antibodies. An anti-tubulin antibody was used as a control of pro-
tein loading. Cells were also exposed to 0.1% DMSO (−) for 12 h, as a con-
Juniperus communis berries extracted with MeOH were subjected
to Kupchanʼs methodology [11] to give n-hexane, CHCl , n-BuOH
3
extracts, and an aqueous residue. The pure compounds were iso-
lated, and their structures were determined by spectroscopic
methods by comparison with the spectral data reported in the lit-
erature: cis-communic acid (6) [15], imbricatolic acid [15-hy-
droxy-labd-8(17)-en-19-oic acid] (2) [16], imbricatolal (3) [17],
junicedric acid (4) [18], junicedral (5), agathadiol (8) [19], isoaga-
tholal (9) [20], myrcecommunic acid (10) [21], sandarocopimaric
acid (11) [22], isopimaric acid (12) [23], oplopanone (16) [24],
6
trol. C 5 × 10 cells were plated in 6-mm wells in the presence or absence of
10 µM imbricatolic acid (IA) for 24 h. Growing cells were incubated with
0.1% DMSO. Cells were resuspended in 300 µL of cold PBS and fixed with
1 mL of 70% ice-cold ethanol for 2 h. FACS analysis was performed as de-
scribed in Materials and Methods by using a DAKA Cytomatio flow cytom-
eter. Data were analyzed using Summit® 4.3 software. All the experiments
were performed in triplicate.
"
and matairesinol (17) [25] (l Fig. 1).
The new dihydrobenzofuran lignan glycoside, named junipero-
2
5
side A (1) ([α]
D
+ 34.4), showed a pseudomolecular ion at m/z
revealed proton signals due to two oxygenated methylene pro-
4
93.2074 in its HRFABMS, and the presence of 25 carbon atoms
tons (δ 3.88 ovl/3.77 dd and 3.58 t), an oxygenated methine sig-
H
1
3
"
in the C NMR spectrum (l Table 2) suggested the molecular for-
mula C25H32O10. In the ESI‑MS (pos. ion mode) spectrum the
pseudomolecular ion peak at m/z 515 [M + Na] was also present.
Its IR spectrum showed absorption bands at 3451 and 1660 cm−
nal at δH 5.57 (d), and one methoxyl signal at δ 3.83 (s). By the
H
1
1
aid of a H- H COSY experiment, a propanol side chain was de-
tected; a second spin system sequence evidenced oxygenated
methylene protons (3.88/3.77) which were connected to a meth-
+
1
corresponding to hydroxyl and aromatic groups, respectively.
ine proton at δH 3.47. This last proton was also coupled with a
1
13
13
The data from H and C NMR spectra of (1) were similar to those
of clemastanin A [26], a glucoside isolated from Clematis species.
proton signal at δ 5.57. In addition to C NMR assignment, fur-
H
ther HSQC experiment correlated the proton resonances of agly-
cone, with the relevant carbons indicating a dehydrodiconiferyl
alcohol-type lignan. Significant HMBC correlations were reported
1
Differences were detected in the sugar moiety. The H NMR spec-
trum of juniperoside A showed three aromatic proton signals at
δH 6.94, 7.06, and 7.08 which were assigned to three protons in
a 1,2,4-trisubstituted benzene ring. Further two aromatic proton
"
in l Table 2. The presence of a sugar moiety was deduced from
1
the anomeric proton signal at δH 5.35 (br s) in the H NMR spec-
signals at δH 6.59 and 6.62 were assigned to two protons in a
trum. Starting from the anomeric proton signal, the proton reso-
nances were assigned by H- H COSYand TOCSY experiments. On
1
1
1
1,3,4,5-tetrasubstituted benzene ring. The H NMR spectrum also
De Marino S et al. Imbricatolic Acid from… Planta Med 2011; 77: 1822–1828

1
826 Original Papers
Fig. 3 Cell lysates were obtained from CaLu-6 cells exposed to 10 µΜ im-
bricatolic acid (IA) for the indicated times A or preincubated with
PD098059 before the stimulation with 10 µΜ IA for 12 h (B and C). Forty
micrograms of proteins were resolved by electrophoresis on 12% SDS-PAGE
and transferred to a nitrocellulose membrane. ERKs phosphorylation was
detected by using an anti-phosphoERK antibody (A and B). The arrows in-
7
dicate the phosphorylated forms of p44MAPK (ERK1-P) and p42MAPK (ERK2-P).
Fig. 4 A Assay of nonradioactive detection of PKC. 1 × 10 CaLu-6 cells
C p21Cip1/Waf1/Sdi1 (p21) was immunodetected by using a specific antibody.
were incubated with 10 µM imbricatolic acid (IA) for the indicated times or
with 0.1% DMSO as a control (−). Cell lysates were purified on DEAE cellu-
lose, and PKC assays were performed in triplicate as described in Materials
and Methods. The reactions were stopped by heating to 95°C for 10 min,
and the samples were separated on a 0.8% agarose gel at 100 V for 15 min
and photographed on a transilluminator. B Membranes were purified from
CaLu-6 cells stimulated with 10 µM imbricatolic acid (IA) for the indicated
times, and PKCδ was detected by a specific antibody. The same filter was
incubated with anti-EGFR antibody as a control of protein loading. Cells
were also exposed to 0.1% DMSO (−) for 12 h. Data is representative of
three independent experiments. C and D Cellular extract were obtained
from CaLu-6 cells stimulated for 12 h with 10 µM imbricatolic acid in the
presence or absence of rottlerin. C Twenty micrograms of proteins were
subjected to immunoblotting analysis with an anti-phosphoERK antibody
An anti-tubulin antibody was used as a control of protein loading. Cells were
also exposed to 0.1% DMSO (−) for 12 h. The experiments were performed
in triplicate.
acid hydrolysis with 2N CF CO H, 1 afforded rhamnose. The L
3
2
configuration of rhamnose was assigned by GC analysis [12].
The connection between the rhamnopyranosyl unit to C-3′ of the
aglycone was verified by the cross-peak between δH 5.35 (H-1′′)
and δ 146.0 (C-3′) in the HMBC experiment. HMBC cross-peaks
C
also revealed the attachment of the methoxyl group at C-3 and
the propanol side chain at C-1′ position. The stereochemistry be-
tween the hydroxymethyl and the aryl group was suggested to be
trans by the ROESY experiment, by a coupling constant of H-7/H-
(
α-pERK). D Forty micrograms of proteins were electrophoresed on 12%
Cip1/Waf1/Sdi1
SDS-PAGE, and the blot was incubated with an anti-p21
(α-p21). The same filters were reprobed with an anti-tubulin antibody. The
experiments were performed in triplicate.
antibody
8
(J = 5.7 Hz) as well as their chemical shifts. An intense ROE was
observed between H-8/H-6′ (δH 3.47/6.62) and H-7/H -9 (δH
2
5
7
.57/3.88–3.77), H-7/H-6 (δH 5.57/6.94), and H-7/H-2 (δH 5.57/
.06). Then the structure of juniperoside A 1 was determined to
ulate the activities of cyclin D-, E-, and A-CDK complexes [9],
which are involved in G1, G1/S, and S checkpoints, respectively.
Labdane treatment of cells resulted in significant time-regulated
be 3-methoxy-3′,4,9,9′-tetrahydroxy-4′,7-epoxy-5′,8-lignan-3′-
O-α-L-rhamnopyranoside.
"
Some labdane-type diterpenes have been shown to inhibit cell
proliferation in several tumor cell lines which express functional
p53 or that do not express p53 [8]. Therefore, we examined the
effect of imbricatolic acid (IA) (2) on cell cycle inhibitory proteins
decrease in the expression of cyclin A, D1, and E1 (l Fig. 2B). Fur-
thermore, DNA staining by propidium iodide showed that the ex-
posure to 10 µM IA for 24 h induces the accumulation in G1 phase
"
(l Fig. 2C). We also analyzed the effects of junicedric acid (4) and
Cip1/Waf1/Sdi1
Kip1
INK4a
p21
, p27 , and p16
, as well as on cell cycle ar-
agathadiol (8), which differ from IA for a carboxylic function on
C15 (4) and for a primary alcoholic function on C19 and a double
bond on C13–C14 (8). No effects of the two compounds on cell
cycle at 10 µM were detected (data not shown).
rest, in p53-null human cancer CaLu-6 cells [27]. Cells were incu-
bated with 10 µM IA for increasing times, and Western blot anal-
ysis showed a significant induction of these CKIs in a time-de-
pendent manner with a maximal accumulation occurring after
In different cell types, the Ras/MAPK pathway mediates growth
"
12 h of treatment (l Fig. 2A). We next evaluated the effect of IA
arrest by controlling cell cycle regulatory proteins [28–31]. Fur-
Cip1/Waf1/Sdi1
on the protein level of cyclins. CKI of the Cip/Kip family can mod-
thermore, in p53-null cells the accumulation of p21
is
De Marino S et al. Imbricatolic Acid from… Planta Med 2011; 77: 1822–1828

Original Papers 1827
Fig. 5 A Cells were incubated in complete culture medium containing
0 µM imbricatolic acid or 0.1% DMSO (−) for the indicated times. MTT solu-
representative of at least three separate experiments of identical design.
1
C and D CaLu-6 cells were exposed to 10 µM imbricatolic acid (IA) for the
indicated times. Fifty micrograms of whole lysates were resolved on 10%
SDS-PAGE and then incubated with specific antibodies against Caspase3 (C)
or PARP1 (D). Cells were also exposed to etoposide as a positive control (PC)
or to 0.1% DMSO (−) for 12 h. An anti-tubulin antibody was used as a control
of protein loading. The experiments were performed in triplicate.
tion was added, and formazan crystals were then dissolved and measured at
λ540 nm. Four independent experiments were performed. B Cells were in-
cubated with 1 µM or 10 µM imbricatolic acid (IA) for the indicated times in
the presence or absence of 1 µM rottlerin for 1 h. Growing cells were exposed
to 0.1% DMSO as a control. Cells were incubated in a solution containing
5
0 µg/mL propidium iodide and analyzed by cytofluorimetry. The data are
a consequence of the activation of Ras-ERK pathway [32]. There-
To evaluate the effect of IA on cell viability of human CaLu-6 cells,
we performed an MTT assay. The cells, exposed for different
times with IA, showed a significant time-dependent inhibition
fore, we investigated the molecular mechanisms underlying the
Cip1/Waf1/Sdi1
upregulation of p21
by analyzing the ability of IA to ac-
"
tivate ERKs. The incubation for increasing times with the labdane
of cell viability (l Fig. 5A), as observed by a 89.8, 53.4, 41.4, 49.8,
"
induced a sustained phosphorylation of ERKs (l Fig. 3A) which
and 62.7% decrease after 3, 12, 24, 48, and 72 h, respectively. To
test whether the decrease in cell growth was due to induction of
apoptosis, we performed propidium iodide staining on CaLu-6
cells exposed for different times or concentrations to IA in the
presence or absence of rottlerin. The results showed that the incu-
"
was prevented by the MEK inhibitor PD098059 (l Fig. 3B). We
also observed that the preincubation with PD098059 prevents
Cip1/Waf1/Sdi1
"
the IA-induced p21
accumulation (l Fig. 3C), suggest-
ing that this requires ERKs activation.
PKCδ is generally considered a growth inhibitor that can contrib-
ute to the p53-independent accumulation of p21Cip1/Waf1/Sdi1 [33,
"
bation with 1 µM or 10 µM IA for 24, 48, or 72 h (l Fig. 5B, panels
"
a–e) as well as the incubation with 1 µM rottlerin (l Fig. 5B, pan-
3
4]. We first investigated the ability of IA to activate PKC in Ca-
el f), or the pretreatment with rottlerin before IA stimulation for
24 h (l Fig. 5B, panel g) did not induce any significant apoptosis.
"
"
Lu-6 cells. l Fig. 4A shows that PKC activity was detectable after
3
h and was sustained after 24 h of exposure to 10 µM IA. We next
Furthermore, we performed Western blot analysis of PARP-1 and
caspase3 by using specific antibodies. l Fig. 5C shows that IA did
"
analyzed PKCδ activation by analyzing cellular partitioning of this
isoenzyme [13]. In response to the labdane, PKCδ translocated in-
to the membrane fraction, and a significant increase in the
not induce the cleavage of the 32 kDa precursor of caspase3 and,
as a consequence, the 116 kDa PARP1 protein resulted not
"
"
amount was observed within 3 h of treatment (l Fig. 4B). We al-
cleaved upon IA treatment (l Fig. 5D). Taken together these data
so observed that preincubation of CaLu-6 cells with 1 µM rottler-
suggest that imbricatolic acid has not an apoptotic effect on CaLu-
6 cells. The same results were obtained on the p53-null PC-3 cell
line (data not shown).
in, a selective inhibitor of PKCδ enzyme activity [35], caused a sig-
"
nificant inhibition of IA-induced ERKs activation (l Fig. 4C) and
Cip1/Waf1/Sdi1
"
p21
accumulation (l Fig. 4D). These results strongly
In summary, we found that imbricatolic acid induces cell cycle ar-
suggest that, in CaLu-6 cells, IA induces PKCδ activation which is
involved in the regulation of ERKs and, in turn, in the accumula-
tion of a specific CKI.
rest in CaLu-6 cells. A possible mechanism is: i) the accumulation
Cip1/Waf1/Sdi1
of p21
, mediated by PKCδ activation and ERKs phos-
phorylation; and ii) the decrease of cyclins A, D1, and E1 levels,
De Marino S et al. Imbricatolic Acid from… Planta Med 2011; 77: 1822–1828

1
828 Original Papers
1
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Conflict of Interest
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