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2005;65:1035-1044. Cancer Res
Dongsool Yim, Rana P. Singh, Chapla Agarwal, et al.
Apoptosis in Human Prostate Carcinoma Cells
A Novel Anticancer Agent, Decursin, Induces G1 Arrest and
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A Novel Anticancer Agent, Decursin, Induces G
1
Arrest and
Apoptosis in Human Prostate Carcinoma Cells
Dongsool Yim,1Rana P. Singh,2Chapla Agarwal,2Sookyeon Lee,1Hyungjoon Chi,4
and Rajesh Agarwal2,3
1Department of Pharmacy, Sahm Yook University, Seoul, Korea; 2Department of Pharmaceutical Sciences, School of Pharmacy and
3University of Colorado Cancer Center, University of Colorado Health Sciences Center, Denver, Colorado; and 4Natural Product
Research Institute, Seoul National University, Seoul, Korea
Abstract
We isolated a coumarin compound decursin (C
19
H
20
O
5
;
molecular weight 328) from Korean angelica (Angelica gigas)
root and characterized it by spectroscopy. Here, for the first
time, we observed that decursin (25-100 Mmol/L) treatment for
24 to 96 hours strongly inhibits growth and induces death in
human prostate carcinoma DU145, PC-3, and LNCaP cells.
Furthermore, we observed that decursinol [where (CH
3
)
2
-
C=CH-COO- side chain of decursin is substituted with -OH]
has much lower effects compared with decursin, suggesting a
possible structure-activity relationship. Decursin-induced
growth inhibition was associated with a strong G
1
arrest
(P< 0.001) in DU145 and LNCaP cells, and G
1
, S as well as G
2
-M
arrests depending upon doses and treatment times in PC-3
cells. Comparatively, decursin was nontoxic to human prostate
epithelial PWR-1E cells and showed only moderate growth
inhibition and G
1
arrest. Consistent with G
1
arrest in DU145
cells, decursin strongly increased protein levels of Cip1/p21 but
showed a moderate increase in Kip1/p27 with a decrease in
cyclin-dependent kinases (CDK); CDK2, CDK4, CDK6, and cyclin
D1, and inhibited CDK2, CDK4, CDK6, cyclin D1, and cyclin E
kinase activity, and increased binding of CDK inhibitor (CDKI)
with CDK. Decursin-caused cell death was associated with an
increase in apoptosis (P< 0.05-0.001) and cleaved caspase-9,
caspase-3, and poly(ADP-ribose) polymerase; however, pre-
treatment with all-caspases inhibitor (z-VAD-fmk) only par-
tially reversed decursin-induced apoptosis, suggesting the
involvement of both caspase-dependent and caspase-indepen-
dent pathways. These findings suggest the novel anticancer
efficacy of decursin mediated via induction of cell cycle arrest
and apoptosis selectively in human prostate carcinoma cells.
(Cancer Res 2005; 65(3): 1035-44)
Introduction
Prostate cancer is the most common cancer as well as the second
leading cause of cancer-related deaths in men in Western countries
(1). One out of nine men over 65 years of age is frequently diagnosed
with prostate cancer in the United States (1, 2). Dietary pattern has
been identified as one of the major factors for the difference in
prostate cancer incidence between Western and Asian countries
(2–4). At present, there is no effective therapy available for the
treatment of androgen-independent stage of prostate cancer, which
usually arises after hormonal deprivation/ablation therapy (5).
Cytotoxic chemotherapies or radiotherapy also do not show any
significant improvement in patient condition due to the high
recurrence of apoptosis resistance hormone refractory prostate
cancer, which is responsible for f28,000 deaths per year (1, 6, 7).
The clinical impact of advanced prostate cancer has led to the
exploration of novel treatment modalities as well as anticancer
agents. In this regard, many nutritive and nonnutritive phytochem-
icals with diversified pharmacologic properties have shown
promising responses for the prevention and/or intervention of
various cancers, including prostate cancer (reviewed in refs. 2–4). In
addition, epidemiologic studies, together with extensive basic
laboratory findings, support the potential role of phytochemicals
in the prevention and treatment of prostate cancer (reviewed in
refs. 8–14).
Herbal medicines including conventional and complimentary
medicines are still prevalent, and serve the medicinal needs of a
large population around the world (reviewed in refs. 15, 16),
which could be assessed by the fact that the global herbal
medicine market is currently worth around $30 billion. Herbal
medicine practice can be traced back to the origin of human
culture, having been mentioned in medical protocols such as
traditional Chinese medicine and Indian Ayurvedic herbal
medicine. At present, there is an increased effort for the
isolation of bioactive microchemicals from medicinal plants for
their possible usefulness in the control of various ailments.
Determining molecular structure and mechanisms of action of
bioactive phytochemicals are equally important for providing the
evidence for their efficacy as well as herbal preparations, which
could also potentially lead to the pharmaceutical development of
synthetic or semisynthetic drugs.
Angelica gigas Nakai (Umbelliferae) root has been traditionally
used in Korean folk medicine as a tonic and for treating anemia
and other common diseases (17). There are some reports about
the pharmacologic evaluation of this plant showing antibacterial
and antiamnestic effects, inhibitory effect on acetylcholinesterase,
depression of cardiac contraction, activation of protein kinase C,
and antitumor activity against sarcoma cancer cells (18–21).
Based on the curative potential, efforts have been made to isolate
the active principle from this plant, which led to the isolation of
many coumarin compounds (22). However, there is no report on
the evaluation of anticancer activity of these compounds against
epithelial cancers. In the present investigation, we used the roots
of A. gigas to isolate decursin and decursinol, and for the first
time tested their efficacy against human prostate carcinoma cells.
A structure-activity relationship was observed for these com-
pounds, with decursin showing greater efficacy in inhibiting
growth and causing death of DU145 cells. The anticancer efficacy
of decursin was also evident against human prostate cancer PC-3
Requests for reprints: Rajesh Agarwal, Department of Pharmaceutical Sciences,
School of Pharmacy, University of Colorado Health Sciences Center, 4200 East Ninth
Street, Box C238, Denver, CO 80262. Phone: 303-315-1381; Fax: 303-315-6281; E-mail:
Rajesh.Agarwal@UCHSC.edu.
D2005 American Association for Cancer Research.
www.aacrjournals.org 1035 Cancer Res 2005; 65: (3). February 1, 2005
Research Article
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and LNCaP cells. Furthermore, we investigated the mechanistic
rationale for the observed efficacy of decursin, which showed an
arrest in cell cycle progression and apoptosis induction as the
most likely targets in prostate cancer cells.
Materials and Methods
Plant Material, Isolation and Characterization of Decursin and
Decursinol. The roots of A. gigas Nakai ( family Umbelliferae) were verified
by Professor Emeritus Hyungjoon Chi (one of the authors in the present
manuscript). The extraction and fractionation of air-dried powdered root was
done as reported recently (22). Silica gel column chromatography was used to
isolate decursin and decursinol (22). These compounds were characterized by
nuclear magnetic resonance (Bruker AVANCE 400 NMR spectrometer) and
mass spectroscopy (by Jeol JMS-AX505-WA mass spectrometer) at Natural
Product Research Institute, Seoul National University, Seoul, Korea. The
purified coumarin compounds, decursin (C
19
H
20
O
5
) with a molecular weight
of 328 (Fig. 1A), and decursinol (C
14
H
14
O
4
) with a molecular weight of 246
(Fig. 1B), were dissolved in DMSO as stock solutions, and used directly in the
cell culture treatments.
Cell Line and Reagents. Human prostate carcinoma cell lines DU145,
PC-3 and LNCaP, and nonneoplastic human prostate epithelial cell line
PWR-1E were obtained from American Type Culture Collection (Manassas,
VA). Prostate cancer cells were cultured in RPMI 1640 with 10% fetal bovine
serum (Hyclone, Logan, UT) under standard culture conditions (37jC, 95%
humidified air, and 5% CO
2
). PWR-1E cells were cultured in keratinocyte
growth medium supplemented with 10% fetal bovine serum, 5 ng/mL
human recombinant epidermal growth factor and 0.05 mg/mL bovine
pituitary extract (American Type Culture Collection). RPMI 1640 and other
culture materials were from Life Technologies, Inc. (Gaithersburg, MD).
Anti-Cip1/p21 antibody was from Calbiochem (Cambridge, MA), and anti-
Kip1/p27 antibody was from Neomarkers, Inc. (Fremont, CA). Antibodies to
cyclin-dependent kinase (CDK); CDK2, CDK4, and CDK6, cyclin D1 and E,
and Rb-glutathione S-transferase fusion protein were from Santa Cruz
Biotechnology, Inc. (Santa Cruz, CA). Anti-caspases primary antibodies and
anti-cleaved poly(ADP-ribose) polymerase (PARP) antibody were from Cell
Signaling Technology (Beverly, MA). Anti-total PARP antibody was from BD
PharMingen (San Diego, CA). Histone H1 was from Boehringer Mannheim,
Corp. (Indianapolis, IN). [g-
32
P] ATP (specific activity 3,000 Ci/mmol) and
enhanced chemiluminescence (Amersham) detection system were from
Amersham Corp. (Piscataway, NJ). Other chemicals were obtained in their
commercially available highest purity grade.
Cell Growth and Death Assays. Cells were plated at 5,000 cells/cm
2
in
60 mm plates under the standard culture conditions detailed above and
after 24 hours, fed with fresh medium and treated with DMSO control or 25,
50, and 100 Amol/L decursin or decursinol. After the desired treatment time,
cells were collected by a brief trypsinization, and counted in duplicate with
a hemocytometer using Trypan blue dye to score dead cells. Each treatment
and time point had three independent plates. The representative data
shown in this study were reproducible in three independent experiments.
Cell Cycle Analysis by Flow Cytometry. Prostate cancer cells and
PWR-1E cells were grown in 10% serum condition in their respective culture
mediums as mentioned above. At f30% confluency, cells were treated with
DMSO control or 25, 50, and 100 Amol/L decursin and at the end of desired
treatment time, cells were collected and processed for cell cycle analysis.
Briefly, 0.5 10
5
cells were suspended in 0.5 mL of saponin/propidium iodide
solution [0.3% saponin (w/v), 25 Ag/mL propidium iodide (w/v), 0.1 mmol
EDTA, and 10 Ag/mL RNase (w/v) in PBS], and incubated overnight at 4jCin
the dark. Cell cycle distribution was then analyzed by flow cytometry using
fluorescence-activated cell sorting core facility of University of Colorado
Cancer Center. Finally, the percentage of cells in different phases of cell cycle
were determined by ModFit LT cell cycle analysis software.
Immunoblotting and Immunoprecipitation. DU145 cells were grown
in RPMI 1640 medium and treated with DMSO control or 25, 50, and
100 Amol/L decursin for the desired times. Equal volumes of DMSO
(0.1% v/v) were present in each treatment. Following decursin treat-
ments, cell lysates were prepared in nondenaturing lysis buffer [10 mmol
Tris-HCl (pH 7.4), 150 mmol NaCl, 1% Triton X-100, 1 mmol EDTA,
1 mmol EGTA, 0.3 mmol phenylmethylsulfonyl fluoride, 0.2 mmol sodium
orthovanadate, 0.5% NP40, and 5 units/mL aprotinin]. For lysate
preparation, medium was aspirated and cells were washed twice with
ice-cold PBS followed by incubation in lysis buffer for 20 minutes. Then,
cells were scraped and kept on ice for an additional 30 minutes, and
finally cell lysates were cleared by centrifugation at 4jC for 30 minutes
in a tabletop centrifuge. Protein concentration in lysates was determined
using Bio-Rad detergent-compatible protein assay kit (Bio-Rad Labora-
tories, Hercules, CA) by Lowry method.
For Western blotting, 60 to 80 Ag of protein lysate per sample was
denatured in 2SDS-PAGE sample buffer and subjected to SDS-PAGE on
12% or 16% tris-glycine gel as published recently (23). The separated
proteins were transferred on to nitrocellulose membrane followed by
blocking of membrane with 5% nonfat milk powder (w/v) in TBS
(10 mmol Tris, 100 mmol NaCl, 0.1% Tween 20) for 1 hour at room
temperature or overnight at 4jC. Membranes were probed for the
protein levels of Cip1/p21, Kip1/p27, CDK2, CDK4, CDK6, cyclin D1, and
E using specific primary antibodies followed by peroxidase-conjugated
appropriate secondary antibody, and visualized by enhanced chemilumi-
nescence (Amersham) detection system. Similarly, for apoptotic mole-
cules, cleaved caspase-9, cleaved caspase-3, total PARP and cleaved PARP
were probed using their specific primary antibodies followed by
appropriate secondary antibody and enhanced chemiluminescence
visualization. Membranes were stripped and reprobed with h-actin
primary antibody as a protein loading control.
For CDK inhibitor (CDKI)-CDK binding study, DU145 cells were
treated with 100 Amol/L decursin for 24 hours and whole cell lysates
were prepared. For each sample, 200 Ag protein lysates was cleared with
protein A/G-plus agarose beads (Santa Cruz) for 45 minutes at 4jC.
Cip1/p21 and Kip1/p27 were then immunoprecipitated from protein
lysates using specific antibody (2 Ag) incubation for 6 hours followed by
addition of 25 AL of protein A/G-plus agarose beads and rocking
overnight at 4jC. Immunoprecipitates were washed thrice with lysis
buffer, and samples were boiled in 2sample buffer for 5 minutes
followed by centrifugation. The resulting clear supernatants were
subjected to SDS-PAGE on 16% gel and Western blotting. Membranes
were then probed with and visualized for CDK2, CDK4, and CDK6 as
detailed above.
Kinase Assays. CDK2 and cyclin E-associated histone H1 kinase
activity was determined as described recently (23). Briefly, 200 Agof
protein lysates from each sample was precleared with protein A/G-plus
agarose beads, and CDK2 and cyclin E proteins were immunoprecipi-
tated using anti-CDK2 and anti–cyclin E antibodies (2 Ag) and protein
A/G-plus agarose beads, respectively. Beads were washed thrice with lysis
buffer, and finally with kinase assay buffer [50 mmol Tris-HCl (pH 7.4),
10 mmol MgCl
2
and 1 mmol DTT]. Phosphorylation of histone H1 was
measured by incubating the beads with 30 AL of ‘‘hot’’ kinase solution
[0.25 AL (2.5 Ag) of histone H1, 0.5 AL(5ACi) of g-
32
P-ATP, 0.5 ALof
0.1 mmol ATP, and 28.75 AL of kinase buffer] for 30 minutes at 37jC.
The reaction was stopped by boiling the samples in SDS sample buffer
for 5 minutes. The samples were then subjected to 12% SDS-PAGE, and
the gel was dried and analyzed by autoradiography (23). Similarly, to
determine CDK4, CDK6, and cyclin D1-associated Rb kinase activities,
these proteins were immunoprecipitated using specific antibodies, and
beads conjugated with antibody and proteins were washed thrice with
Rb-lysis buffer [50 mmol HEPES-KOH (pH 7.5), 150 mmol NaCl, 1 mmol
EDTA, 2.5 mmol EGTA, 1 mmol DTT, 80 mmol h-glycerophosphate,
1 mmol NaF, 0.1 mmol sodium orthovanadate, 0.1% Tween 20, 10%
glycerol, 1 mmol phenylmethylsulfonyl fluoride, and 10 Ag/mL leupeptin and
aprotonin] and twice with Rb-kinase assay buffer [50 mmol HEPES-KOH
(pH 7.5), 2.5 mmol EGTA, 10 mmol h-glycerophosphate, 1 mmol NaF, 10
mmol MgCl
2
, 0.1 mmol sodium orthovanadate, and 1 mmol DTT].
Phosphorylation of Rb was measured by incubating the beads with 30 AL
of hot Rb-kinase solution (2 Ag of Rb-glutathione S-transferase fusion
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Figure 1. Structure, growth inhibitory, and cell death effects of decursin and decursinol on DU145 cells. Chemical structure of decursin (A) and decursinol (B),
isolated from the roots of A. gigas . To assess the effect of decursin and decursinol on exponentially growing DU145 cells, 5,000 cells/cm
2
were plated in 60 mm dishes
and the next day, cells were treated either DMSO vehicle control or 25, 50, and 100 Amol/L doses of decursin or decursinol in complete medium. After 24, 48, 72,
and 96 hours of these treatments, total cells were counted using a hemocytometer (C), dead cells were scored using Trypan blue dye exclusion method (D).
Mean FSE of three independent plates; each sample was counted in duplicate. DN, decursin; DL, decursinol; *, P < 0.05;
$
,P< 0.01;
#
,P< 0.001 versus control.
A Novel Anticancer Agent Decursin against Prostate Carcinoma Cells
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protein, 5 ACi of g-
32
P-ATP, and 0.1 mmol ATP in Rb-kinase buffer) for
30 minutes at 37jC. Reaction was stopped by boiling the samples in
5SDS sample buffer for 5 minutes. Samples were analyzed by SDS-PAGE,
and the gel was dried and subjected to autoradiography (23).
Apoptotic Cell Death Assay. To quantify decursin-induced apoptotic
death of human prostate carcinoma DU145 cells, annexin V, and propidium
iodide staining was done followed by flow cytometry, as described recently
(23, 24). Briefly, after treatment (DMSO control, 50 and 100 Amol/L decursin
for 24 and 48 hours), cells were collected and subjected to annexin V and
propidium iodide staining using Vybrant Apoptosis Assay Kit2 (Molecular
Probes, Inc., Eugene, OR) and following the step-by-step protocol provided
by the manufacturer. Annexin V binds to phospatidylserine, which is
translocated from inner to outer leaflet of the plasma membrane in
apoptotic cells. In all-caspases inhibitor (z-VAD-fmk; Enzyme Systems
Products, ICN Pharmaceuticals, Inc., Aurora, OH) treatment, cells were
pretreated with the inhibitor for 2 hours before decursin treatment.
Caspase Activity Assay. Caspase-3 activity was assayed by colori-
metric protease assay ApoTarget kit (BioSource International, Inc., CA)
following manufacturer’s protocol as published recently (24). Briefly, at the
end of treatment with either 100 Amol/L dose of decursin, or all-caspases
inhibitor z-VAD-fmk (100 Amol/L), or both, in which cells were pretreated
for 2 hours with the inhibitor for a total of 48 hours, cells were collected and
cell lysates prepared in cell lysis buffer (TBS containing detergent). Protein
lysate (100 Ag per sample) was mixed with 2reaction buffer and 200 Amol/
L substrate (DEVD-pNA for caspase-3) and incubated at 37jC for 3 hours in
the dark. These assay conditions are standardized and products of the
reaction remain in the linear range of detection. Developed color was
measured at 405 nm in microplate reader, and blank reading was subtracted
from each sample reading before calculation.
Statistical Analysis. The data were analyzed using the Jandel Scientific
SigmaStat 2.03 software. Student’s ttest was employed to assess the
statistical significance of difference between control and decursin-treated or
decursinol-treated groups. A statistically significant difference was consid-
ered to be present at P< 0.05. Autoradiograms/bands were scanned with
Adobe Photoshop 6.0 (Adobe Systems, Inc. San Jose, CA).
Results
Nuclear Magnetic Resonance and Mass Characterization of
Decursin and Decursinol. Decursin: white crystals from CH
3
OH.
[
1
H] NMR (400 MHz, CDCl
3
)y
H
(ppm): 7.57 (1H, d, J = 9.5 Hz, H-4),
7.14 (1H, s, H-5), 6.78 (1H, s, H-8), 6.21 (1H, d, J = 9.5 Hz, H-3), 5.64
(1H, s, H-2V), 5.06 (1H, t, J = 4.6 Hz, H-3V), 3.17 (1H, dd, J = 16.0, 4.2
Hz, H-4V
a
), 2.85 (1H, dd, J = 16.0, 4.2 Hz, H-4V
b
), 2.12 (3H, s, 3V-CH
3
),
1.86 (3H, s, H-400), 1.36 (3H, s, gem-CH
3
), 1.34 (3H, s, gem-CH
3
). Fast
atom bombardment mass spectroscopy: m/z(rel. int., %): 329 (100)
[M + H]
+
, 228 (100), 213 (95), 154 (45), 136 (45; Fig. 1A). Decursinol:
white crystals from CH
3
OH. [
1
H] NMR (400 MHz, CDCl
3
)
H
(ppm):
7.57 (1H, d, J = 9.5 Hz, H-4), 7.17 (1H, s, H-5), 6.77 (1H, s, H-8), 6.21
(1 H, d, J = 9.2 Hz, H-3), 3.86 (1H, t, J = 4.8 Hz, H-3V), 3.10 (1H, dd,
J = 17.0, 4.5 Hz, H-4V
a
) 2.83 (1H, dd, J = 17.0, 4.5 Hz, H-4V
b
) 1.39 (3H, s,
gem-CH
3
), 1.36 (3H, s, gem-CH
3
). Fast atom bombardment mass
spectroscopy: m/z(rel. int., %): 247 (100) [M + H]
+
, 246 (85), 154
(100), 136 (90; Fig. 1B).
Effects of Decursin and Decursinol on Growth and Death of
DU145 Cells. To assess the biological activity of these com-
pounds in terms of cell growth and death, DU145 cells were
treated with 25, 50, and 100 Amol/L doses of decursin and
decursinol for 24, 48, 72, and 96 hours. Decursin (25-100 Amol/L)
showed a strong dose- and time-dependent inhibition of cell
growth, accounting for 22% to 51% (P< 0.05-0.001), 21% to 68%
(P< 0.01-0.001), 9% to 72% (P< 0.001), and 42% to 90%
(P< 0.001) growth inhibition after 24, 48, 72, and 96 hours of
treatment, respectively (Fig. 1C). However, similar treatment with
decursinol (25-50 Amol/L for 24, 48, 72, and 96 hours) showed
almost half the efficacy of decursin, accounting for 11% to 36%
(P< 0.05-0.001), 9% to 45% (P< 0.01-0.001), 9% to 27% (P< 0.001),
and 31% to 51% (P< 0.001) growth inhibition, respectively
(Fig. 1C). These data suggest that decursin is much more potent
in inhibiting the growth of DU145 as compared with decursinol.
The differences in biological activity of these two compounds
could be attributed to the difference in their chemical structures
(Fig. 1Aand B). The increased activity of decursin could be, most
likely, due to (CH
3
)
2
-C=CH-COO- side chain of decursin which
is substituted with OH group in decursinol (Fig. 1Aand B).
Furthermore, we observed that both compounds have cytotoxic
effect on DU145 cells, in which similar treatment (25-100 Amol/L
for 24, 48, 72, and 96 hours) with decursin caused 15% to 45%
(P< 0.05-0.001) cell death versus 11% to 12% in controls (Fig. 1D),
whereas decursinol caused 13% to 32% (P< 0.05-0.001) cell death
as compared with 8% to 13% in controls (Fig. 1D). When these
cytotoxicity data were compared between these two agents, at
similar doses and treatment times, decursin showed a relatively
better cell death effect compared with decursinol, except at
48 hours of treatment; however, it was statistically significant
(decursin versus decursinol) only at 25 Amol/L dose for 96-hour
(P< 0.05) and 100 Amol/L dose for 72-hour (P< 0.001)
treatments. These data suggest a relatively higher cytotoxic effect
of decursin as compared with decursinol, and further support a
possible structure-activity relationship in their overall effects in
DU145 cells. Next, we investigated the growth inhibitory and
cytotoxic effects of decursin on human prostate cancer PC-3 and
LNCaP cells, and human prostate epithelial PWR-1E cells, and
mechanisms associated with these effects of decursin in DU145
cells.
Effect of Decursin on Growth and Death of Human
Prostate Cancer PC-3 and LNCaP Cells, and Human Prostate
Epithelial PWR-1E Cells. As compared with DU145 cells,
decursin (25-100 Amol/L) treatment of PC-3 cells for 24, 48, 72,
and 96 hours, showed stronger dose- and time-dependent growth
inhibitory effect accounting for 7% to 54% (P< 0.05-0.001), 25% to
75% (P< 0.05-0.001), 36% to 84% (P< 0.05-0.001), and 49% to 91%
(P< 0.05-0.001) inhibition, respectively (Fig. 2A). However, cytotoxic
effect of decursin in PC-3 cells was limited to higher doses only,
showing 3% to 18% (P> 0.05 to < 0.05-0.001) dead cells as compared
with 2% to 5% in controls (Fig. 2A). The growth inhibitory effect of
decursin in LNCaP cells was stronger than both DU145 and PC-3
cells. In similar decursin treatment for 24, 48, 72, and 96 hours, 9% to
72% (P> 0.05 to < 0.005-0.001), 48% to 83% (P< 0.005-0.001), 76% to
94% (P< 0.001), and 79% to 98% (P< 0.001) cell growth inhibition was
observed in LNCaP cells, respectively (Fig. 2B). The cytotoxic effect
of decursin in LNCaP cells was also greater than that of DU145 and
PC-3 cells accounting for 16% to 52% (P> 0.05 to < 0.05-0.001) cell
death as compared with 8% to 16% in controls (Fig. 2B). Overall,
these results suggested growth inhibitory and cytotoxic effects of
decursin in human prostate carcinoma PC-3, LNCaP as well as
DU145 cells.
Furthermore, using nonneoplastic human prostate epithelial
PWR-1E cells, we assessed whether decursin has any differential
sensitivity to normal versus cancer cells. Surprisingly, we did not
observe any increase in dead cells following decursin treatment;
it was even significantly lower (P< 0.05 versus control) following
72 hours of treatment at the 100 Amol/L dose (Fig. 2C). A
decrease in cell number, however, was observed after decursin
treatment of PWR-1E cells, but it was not as strong as in the
case of prostate carcinoma cells (Fig. 2C). For instance, growth
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inhibition caused by 50 to 100 Amol/L doses of decursin was
56% to 75% (48 hours) and 64% to 84% (72 hours) in PC-3 cells;
and 58% to 83% (48 hours) and 77% to 94% (72 hours) in LNCaP
cells as compared with 14% to 62% (48 hours) and 41% to 77%
(72 hours) in PWR-1E cells (Fig. 2). After 24 hours of decursin
treatment, we did not observe any considerable growth
inhibition or cell death in PWR-1E cells (data not shown).
These results suggest that decursin is not toxic to prostate
epithelial cells; however, it inhibits their growth with lesser
efficacy as compared with cancer cells in culture.
Decursin Induces a Strong G
1
Arrest in Cell Cycle
Progression of Prostate Cancer Cells. Inhibition of deregulated
cell cycle progression in cancer cells is an effective strategy to
halt tumor growth (14, 25). Because we observed a strong growth
Figure 2. Growth inhibitory and cell death effects of decursin on PC-3, LNCaP, and PWR-1E cells. To assess the effect of decursin on exponentially growing
human PCA PC-3 (A), LNCaP (B) cells, and nonneoplastic prostate epithelial PWR-1E cells (C), 5,000 cells/cm
2
were plated in 60 mm dishes and the next day, cells
were treated either DMSO vehicle control or 25, 50, and 100 Amol/L doses of decursin in complete medium. After the indicated treatment times, total cells were
counted using a hemocytometer, and dead cells were scored using Trypan blue dye exclusion method. Mean FSE of three independent plates; each sample was
counted in duplicate. DN, decursin; DL, decursinol; *, P < 0.05;
$
,P< 0.01;
#
,P< 0.001 versus control.
A Novel Anticancer Agent Decursin against Prostate Carcinoma Cells
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inhibitory effect of decursin, we then analyzed its possible
inhibitory effect on cell cycle progression following 25, 50, and
100 Amol/L doses of decursin treatment for 12 to 96 hours. We
observed that decursin induces G
1
arrest, which started as early
as 12 hours after the treatment in DU145 cells (data not shown).
An optimum dose-dependent effect was observed at 24 hours of
treatment where decursin caused 52%, 65%, and 78% DU145 cells
in G
1
phase (P< 0.001) as compared with 45% in control (Fig. 3A
and B); this effect started diminishing after 48 hours of treatment
(data not shown). An increase in G
1
cell population was mostly
at the expense of S phase cells (P< 0.05-0.01) with a minimal
decrease in G
2
-M cell population (Fig. 3B).
Similarly, in PC-3 cells, a strong G
1
arrest was observed at 25 and
50 Amol/L decursin treatment for 24 hours (Fig. 4A), which started
diminishing at later treatment times leading to a moderate G
2
-M
arrest (data not shown). However, 100 Amol/L dose of decursin
invariably showed an increase in S and/or G
2
-M phase cell
population in PC-3 cells at all treatment times (Fig. 4A; data for
later treatment times not shown). Similar to DU145 and PC-3 cells,
decursin (25-50 Amol/L) caused significant increase in G
1
cell
population at the expense of S as well as G
2
-M phase cell
populations in LNCaP cells (Fig. 4B). However, in contrast to
DU145 and PC-3 cells, this effect of decursin in LNCaP cells did
not diminish and remained sustained even at 96 hours of the
treatment (data not shown). Higher doses (100 Amol/L) of
decursin, interestingly, showed a time-dependent increase in G
1
arrest in LNCaP cells (data not shown). Furthermore, in
nonneoplastic prostate epithelial PWR-1E cells, only a moderate
increase in G
1
arrest was observed only at higher doses (100
Amol/L) of decursin treatment (Fig. 4C). However, both lower
doses (25 and 50 Amol/L) that were effective in prostate cancer
cells did not show any cell cycle effect in PWR-1E cells (Fig. 4C;
and data not shown). These results suggest that inhibition of
deregulated cell cycle progression could be one of the molecular
events associated with selective anticancer efficacy of decursin in
prostate cancer cells.
Decursin Up-regulates Cip1/p21 and Kip1/p27 Levels, and
Inhibits CDK- and Cyclin-Associated Kinase Activity. Since,
we observed optimum G
1
cell cycle arrest by decursin at 24 hours
in DU145 cells, and a relatively similar effect at 48 hours (data
not shown), total cell lysates were prepared following decursin
treatment of cells at 25, 50, and 100 Amol/L doses for 24 and
48 hours. Western blot analysis of total cell lysates (80 Ag/sample)
showed a strong dose-dependent increase in the expression of
Cip1/p21, which was moderate in case of Kip1/p27 (Fig. 5A).
Reprobing of membranes for h-actin confirmed equal protein
loading (Fig. 5A,bottom). In the studies analyzing its effect on
the levels of CDKs and cyclins associated with G
1
phase,
decursin treatment for 24 hours did not show any alteration in
protein levels of CDK2, CDK4, CDK6, cyclin D1, and cyclin E;
however, 48 hours of treatment showed a dose-dependent
decrease in the expression of these proteins except cyclin E
(Fig. 5B).
Next, cell lysates were assayed for CDK2, CDK4, CDK6, cyclin D1,
and cyclin E kinase activity. As shown in Fig. 5C, compared with
control, decursin treatment resulted in almost complete inhibition
in CDK2, cyclin E, and cyclin D1 kinase activity; however, the
inhibitory effect of decursin on CDK4- and CDK6-associated kinase
activity was of lower magnitude (Fig. 5C). We anticipated that the
observed inhibition in kinase activity of these CDKs and cyclins
could in part be due to an up-regulated level of CDKIs and their
binding with CDK-cyclin complex, causing a resultant G
1
arrest. To
support this anticipation, we immunoprecipitated Cip1/p21 and
Kip1/p21 from total cell lysates, and studied their binding with
CDK2, CDK4, and CDK6, which showed an increase in the bound
levels of these proteins following decursin treatment (Fig. 5D).
These results suggest a possible increase in CDK-cyclin-CDKI
complex formation accompanied by a decrease in CDK kinase
activity following decursin treatment.
Decursin Induces Apoptotic Death of DU145 Cells. During
cell growth assay, we observed that decursin causes a significant
increase in DU145 cell death, where higher doses and longer
treatment times were more effective. Here, using annexin
V-staining and flow cytometry, we assessed whether decursin-
caused cell death is mediated by apoptosis. Cells were treated with
DMSO control, 50, and 100 Amol/L doses of decursin for 24 and 48
hours for the apoptosis analysis. Our data show a dose-dependent
increase (up to f3-fold, P< 0.05-0.001) in apoptotic cell population
following decursin treatment (Fig. 6A). Lower doses (50 Amol/L) of
Figure 3. Effect of decursin on cell cycle progression in DU145 cells. Cells were cultured in complete medium, and treated with either DMSO vehicle control or 25
to 100 Amol/L doses of decursin. After 24 hours of these treatments, cells were collected, washed with PBS, digested with RNase, and then cellular DNA was
stained with propidium iodide as detailed in Materials and Methods. Flow cytometric analysis was then performed for cell cycle distribution. A, propidium iodide
fluorescence pattern for cell cycle distribution in different treatments. B, the percentage of cell cycle distribution data for each treatment group. Mean FSE of three
independent samples. DN, decursin; *, P < 0.05;
$
,P< 0.01;
#
,P< 0.001 versus control.
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decursin were not as effective as higher doses (100 Amol/L) which
showed more apoptotic cell death (23%) at 48 hours as compared
with 24 hours of treatment (10%) versus controls showing 5% to 8%
apoptotic cell death (Fig. 6A). These data suggest that apoptosis
induction could be a major mechanism of decursin-caused death of
prostate cancer cells. With similar decursinol treatment, we did not
observe any considerable apoptosis induction in DU145 cells (data
not shown).
Role of Caspase Activation in Decursin-Caused Apoptosis.
Activation of caspase cascade leading to PARP cleavage is
regarded as a major pathway in apoptosis induction (26). Based
on the above results showing induction of apoptosis by decursin,
we analyzed the levels of cleaved caspase-9 and caspase-3
following 48 hours of treatment. Cleavage of caspases is directly
related to their activation status. Our data show that decursin
caused an increase in cleaved caspase-9 and caspase-3, which
were very prominent at 100 Amol/L dose of decursin (Fig. 6B,
rows 1 and 2). Consistent with the cleavage of caspases, decursin
also caused a strong increase in PARP cleavage (Fig. 6B). First,
we used PARP antibody (BD PharMingen) which recognizes both
total (116 kDa) as well as cleaved PARP (89 kDa; Fig. 6B,row 3);
and later the same membrane was stripped and reprobed with
specific cleaved PARP antibody (Cell Signaling) which showed
better sensitivity for the detection of cleaved PARP fragment
(Fig. 6B,row 4). Furthermore, these membranes were also
checked for the level of h-actin as a loading control; only a
representative blot is shown in Fig. 6B(row 5).
To further establish the role of caspase activation in decursin-
caused apoptosis, we used all-caspases inhibitor z-VAD-fmk
(2 hours pretreatment), which only partially reversed the
decursin-induced apoptosis (Fig. 6C). To confirm whether the
dose of all-caspases inhibitor used in the study was sufficient to
inhibit caspase activity, caspase-3 activity was also measured under
identical treatments. As shown in Fig. 6D, 100 Amol/L dose of
caspase inhibitor completely inhibited 100 Amol/L decursin-
induced (f3 fold) caspase-3 activity. Overall, these results
suggested the involvement of both caspase-dependent and
caspase-independent pathway(s) in decursin-induced apoptotic
death of DU145 cells.
Discussion
The central and novel finding in the present study is the
identification of in vitro anticancer efficacy of decursin against
advanced human prostate carcinoma DU145, PC-3, and LNCaP
cells. The completed studies clearly and convincingly show that
decursin causes a G
1
cell cycle arrest via an induction of Cip1/p21
and to a lesser extent Kip1/p27 together with an inhibition in CDK-
cyclin kinase activity as an underlying mechanism in its DU145 cell
growth inhibition. Furthermore, this agent causes apoptosis
induction involving both caspase-dependent and caspase-indepen-
dent mechanisms in DU145 cells. More importantly, decursin was
nontoxic to nonneoplastic human prostate epithelial cells and
showed only a moderate cell growth inhibition with a slight effect
on cell cycle progression.
The present study also provides an evidence for the structure
and anticancer activity of the coumarin compounds decursin
and decursinol against hormone refractory human prostate
cancer DU145 cells. Based on structural analysis, the side chain
in decursin, which is substituted with an -OH group in
decursinol (Fig. 1), could be attributed to the enhanced
anticancer efficacy of decursin. We also observed that decursi-
nol-caused death of DU145 cells was not associated with
apoptosis induction (data not shown); therefore, the substituted
side chain in decursin might be solely responsible for its
apoptotic efficacy in DU145 cells. Overall, the differential efficacy
of these compounds in DU145 cells was in accord with their
structure-activity relationship in inhibiting acetylcholinesterase
activity reported earlier (18).
CDKs, CDKIs, and cyclins play essential roles in the regulation of
cell cycle progression (25). CDKIs are tumor suppressor proteins
that down-regulate the cell cycle progression by binding with active
CDK-cyclin complexes and thereby inhibiting their kinase activities
Figure 4. Effect of decursin on cell cycle progression in PC-3, LNCaP, and
PWR-1E cells. Cells were cultured in complete medium, and treated with either
DMSO vehicle control or 25 to 100 Amol/L doses of decursin. After 24 hours of
these treatments, cells were processed and analyzed by flow cytometry as
mentioned in Fig. 3. The percentage of cell cycle distribution data for PC-3 (A),
LNCaP (B), and PWR-1E (C) cells are shown as mean FSE of three
independent samples. DN, decursin; *, P < 0.05;
$
,P< 0.01;
#
,P< 0.001 versus
control.
A Novel Anticancer Agent Decursin against Prostate Carcinoma Cells
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(25, 27, 28). Cip1/p21 is a universal inhibitor of CDKs whose
expression is mainly regulated by the p53 tumor suppressor protein
(29); however, Kip1/p27 is up-regulated in response to antiprolifer-
ative signals (30, 31). The increased expression of G
1
cyclins in
cancer cells provides them an uncontrolled growth advantage
because most of these cells either lack CDKI, harbor nonfunctional
CDKI, or CDKI expression is not at a sufficient level to control
CDK-cyclin activity (28, 32). Consistent with these reports, cell cycle
analysis data showed that decursin caused a strong G
1
arrest in cell
cycle progression of prostate cancer cells. Furthermore, mechanis-
tic investigation showed that decursin-induced G
1
arrest in DU145
cells is mainly mediated via an up-regulation of Cip1/p21 (strong)
and Kip1/p27 (moderate). As CDK activity is essential for driving
the cells through G
1
-S transition (33), concomitant with CDKIs
induction, we also observed an increase in Cip1/p21-CDK2/CDK4/
CDK6 and Kip1/p27-CDK2/CDK4/CDK6 binding together with an
inhibition in CDK-cyclin kinase activity in decursin-treated cells.
The increased expression of CDKIs by decursin might also have a
direct relevance in prostate cancer growth and progression, as
decreased Kip1/p27 expression in prostatic carcinomas has been
associated with aggressive phenotype and poor prognosis, and a
failure of irradiation response in prostate cancer patients has been
linked to the loss of Cip1/p21 function (34). Recent studies have
also indicated the prognostic significance of CDKIs in prostate
cancer (35). Specifically, Kip1/p27 expression has been shown to be
an independent predictor of prostate-specific antigen failure
following radical prostatectomy (36), and its low expression is
correlated with poor disease-free survival in prostate cancer
patients (37).
Most of the presently available cytotoxic anticancer drugs
mediate their effect via apoptosis induction in cancer cells
(26, 38), and apoptosis is suggested as one of the major mech-
anisms for the targeted therapy of various cancers including
prostate cancer (26, 38–40). In case of advanced prostate cancer,
cancer cells become resistant to apoptosis and do not respond to
cytotoxic chemotherapeutic agents (7). Therefore, the agents that
induce apoptotic death of hormone-refractory prostate cancer cells
could be useful in controlling this malignancy (41). Consistent with
this approach, our data showing an induction of apoptotic death of
advanced prostate cancer cells by decursin could be of greater
significance in identifying another anticancer mechanism (together
with cell cycle arrest) of decursin for its possible application in
prostate cancer control. Because induction of CDKIs has been
reported in anticancer agent–caused apoptosis in human prostate
cancer cells (42), the decursin-induced CDKIs could be, in part,
responsible for the observed apoptotic death of DU145 cells. The
increase in the levels of active caspase-9 and caspase-3 as well as
cleaved PARP suggested that caspase activation is another
Figure 5. Effect of decursin on G
1
cell cycle regulators and CDK-associated and cyclin-associated kinase activities in DU145 cells. Cells were cultured in complete
medium, and treated with either DMSO vehicle control or 25 to 100 Amol/L doses of decursin as described in Materials and Methods. Aand B, at the end of treatments,
total cell lysates were prepared and subjected to SDS-PAGE followed by Western immunoblotting. Membranes were probed with anti-Cip1/p21, Kip1/p27, CDK2,
CDK4, CDK6, cyclin D1, cyclin E, and h-actin antibodies followed by peroxidase-conjugated appropriate secondary antibodies, and visualized by enhanced
chemiluminescence detection system. The experiments were repeated thrice with similar results and a representative blot is shown for each protein. C, CDK2
and cyclin E kinase activities were determined by in-bead histone H1 kinase assay using immunoprecipitated CDK2 and cyclin E from total cell lysates (200 Ag protein)
using specific antibodies. CDK4, CDK6, and cyclin D1-associated kinase activities were determined by in-bead RB-GST fusion protein kinase assay using
immunoprecipitated CDK4, CDK6, and cyclin D1 from total cell lysates (200 Ag protein) using specific antibodies as described in Materials and Methods. After the assay,
the labeled substrates were subjected to SDS-PAGE, and the gel was dried and exposed to X-ray film. D, Cip1/p21 and Kip1/p27 were immunoprecipitated using
primary specific antibodies, followed by Western blot analysis for CDK2, CDK4, and CDK6 as detailed in Materials and Methods. IP, immunoprecipitation; IB,
immunoblotting.
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important mechanism in decursin-induced apoptosis in prostate
cancer cells. However, use of all-caspases inhibitor also showed the
involvement of caspase-independent mechanism(s) in decursin-
mediated apoptosis of DU145 cells. Further studies are needed to
explore the caspase-independent mechanism(s) of apoptosis
induction by decursin.
Several studies suggest that hormone refractory, advanced
prostate cancer phenotype is associated with many changes
including deregulated cell cycle and cell survival signaling, and
inactivation of p53 and pRb such as in DU145 cells. Therefore,
findings in the present study have clinical significance in the fact
that decursin caused p53-independent up-regulation of Cip1/p21
as DU145 cells do not have functional p53 gene and are also
mutated for Rb. Another significant observation was that
decursin showed a sustained G
1
arrest in LNCaP cells, which
have functional p53 and Rb genes, although this effect was
diminished with the increase in treatment time in DU145 and
PC-3 cells lacking functional p53 and pRb. This observation
suggests the most likely role of p53 and/or pRb in decursin-
induced G
1
arrest in LNCaP cells; however, more studies are
needed to confirm this anticipation. Furthermore, we also
observed that decursin induces S and/or G
2
-M arrest at the
expense of G
1
phase cell population with the increase in
treatment time or at 100 Amol/L dose only in PC-3 cells and not
in DU145 or LNCaP cells, suggesting differential molecular
determinants of this cell cycle effect of decursin in PC-3 cells.
In conclusion, our present findings showing the in vitro
anticancer efficacy of decursin, with mechanistic rationale (cell
cycle arrest and apoptosis induction), against advanced human
prostate cancer cells without any cytotoxicity to nonneoplastic
prostate epithelial cells, warrant its further in vivo efficacy studies
in preclinical human prostate cancer models as well as estimation
of pharmacologically achievable doses having biological signifi-
cance in in vitro studies. The positive outcomes of such an in vivo
study could form a strong basis for the development of decursin
as a novel agent for human prostate cancer prevention and/or
intervention.
Acknowledgments
Received 6/4/2004; revised 11/17/2004; accepted 11/22/2004.
Grant support: Supported in part by grants NCI RO1 CA91883 and CA102514.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
Figure 6. Apoptotic effect of decursin on DU145 cells. A, cells were cultured in complete medium, and treated with either DMSO vehicle control or 25 to 100
Amol/L doses of decursin as described in Materials and Methods. At the end of treatments, total cells were collected and stained with annexin V/propidium iodide as
mentioned in Materials and Methods followed by flow cytometric analysis. Data are presented as a percentage of annexin V/propidium iodide stained cells for each
treatment. B, after 48 hours of decursin treatments, cell lysates were prepared and SDS-PAGE and Western blot analysis were performed for cleaved caspase-9,
cleaved caspase-3, total and cleaved PARP, and h-actin using specific antibodies as described in Materials and Methods. C, cells were cultured in complete medium,
and treated with either DMSO vehicle control, 100 Amol/L all-caspases inhibitor (z-VAD-fmk), or 100 Amol/L dose of decursin and/or all caspase inhibitor 2 hours prior to
decursin in combination treatment for a total 48 hours. At the end of treatments, cells were analyzed for apoptosis as detailed in Materials and Methods. D, in similar
treatment as in (C), cell lysates were prepared and caspase-3 activity assay was performed as detailed in Materials and Methods. Data presented in (A), (C), and (D)
are mean FSE of three independent samples in each treatment group. CI, 100 Amol/L all caspase inhibitor; DN, 100 Amol/L decursin;
#
,P< 0.05;
$
,P< 0.01; *,
P< 0.001 versus control.
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