Decursin suppresses human androgen-independent PC3 prostate cancer cell proliferation by promoting the degradation of β-catenin

Article abstract of DOI:10.1124/mol.107.040253

Alterations in the Wnt/β-catenin pathway are associated with the development and progression of human prostate cancer. Decursin, a pyranocoumarin isolated from the Korean Angelica gigas root, inhibits the growth of androgen-independent human prostate cancer cells, but little is known about its mechanism of action. Using a cell-based screen, we found that decursin attenuates the Wnt/β-catenin pathway. Decursin antagonized β-catenin response transcription (CRT), which was induced with Wnt3a-conditioned medium and LiCl, by promoting the degradation of β-catenin. Furthermore, decursin suppressed the expression of cyclin D1 and c-myc, which are downstream target genes of β-catenin and thus inhibited the growth of PC3 prostate cancer cells. In contrast, decursinol, in which the (CH₃) ₂-C=CH-COO- side chain of decursin is replaced with -OH, had no effect on CRT, the level of intracellular β-catenin, or PC3 cell proliferation. Our findings suggest that decursin exerts its anticancer activity in prostate cancer cells via inhibition of the Wnt/β-catenin pathway. Copyright

Full text of DOI:10.1124/mol.107.040253

0
026-895X/07/7206-1599–1606$20.00
MOLECULAR PHARMACOLOGY
Copyright © 2007 The American Society for Pharmacology and Experimental Therapeutics
Mol Pharmacol 72:1599–1606, 2007
Vol. 72, No. 6
40253/3277472
Printed in U.S.A.
Decursin Suppresses Human Androgen-Independent PC3
Prostate Cancer Cell Proliferation by Promoting the
Degradation of ␤-Catenin
Gyu-Yong Song, Jee-Hyun Lee, Munju Cho, Byeoung-Soo Park, Dong-Eun Kim, and
Sangtaek Oh
College of Pharmacy, Chungnam National University, Daejeon, Korea (G.-Y.S., J.-H.L.); PharmcoGenomics Research Center,
Inje University, Busan, Korea (M.C., S.O.); Institute of Ecological Phytochemistry, Hankyang National University, Ansung, Korea
(
B.-S.P.); and Department of Bioscience and Biotechnology, Konkuk University, Seoul, Korea (D.-E.K.)
Received July 20, 2007; accepted September 12, 2007
ABSTRACT
Alterations in the Wnt/␤-catenin pathway are associated with degradation of ␤-catenin. Furthermore, decursin suppressed
the development and progression of human prostate cancer. the expression of cyclin D1 and c-myc, which are downstream
Decursin, a pyranocoumarin isolated from the Korean Angelica target genes of ␤-catenin and thus inhibited the growth of PC3
gigas root, inhibits the growth of androgen-independent human prostate cancer cells. In contrast, decursinol, in which the
prostate cancer cells, but little is known about its mechanism of (CH ) –CϭCH–COO– side chain of decursin is replaced with
3
2
action. Using a cell-based screen, we found that decursin –OH, had no effect on CRT, the level of intracellular ␤-catenin,
attenuates the Wnt/␤-catenin pathway. Decursin antagonized or PC3 cell proliferation. Our findings suggest that decursin
␤-catenin response transcription (CRT), which was induced exerts its anticancer activity in prostate cancer cells via inhibi-
with Wnt3a-conditioned medium and LiCl, by promoting the tion of the Wnt/␤-catenin pathway.
Wnts, cysteine-rich secreted glycoproteins, play important signal, casein kinase 1 and glycogen synthase kinase-3␤
roles in cell proliferation, fate determination, and movement (GSK-3␤), which form a complex with adenomatous polyposis
during development (Wodarz and Nusse, 1998; Polakis, coli (APC)/Axin, sequentially phosphorylate ␤-catenin at
2
000). Depending on the type of Wnt and the Frizzled (Fz) Ser33, Ser37, Thr41, and Ser45 (Liu et al., 2002). Phosphor-
receptor it engages, one of three intracellular signaling path- ylated ␤-catenin is then recognized by F-box ␤-transducin
ways may be activated: the Wnt/␤-catenin pathway, the pla- repeat-containing protein (␤-TrCP), a component of the ubiq-
2ϩ
nar cell polarity pathway, and the Wnt/Ca pathway (K u¨ hl
et al., 2000; Winklbauer et al., 2001; Giles et al., 2003).
Among these, the best understood is the Wnt/␤-catenin path-
way, which is activated by the interaction of Wnt1, Wnt3a,
and Wnt8 with Fz receptors and low-density lipoprotein re-
ceptor-related protein 5/6 coreceptors (Giles et al., 2003). The
pathway is regulated mainly by the level of cytosolic ␤-cate-
nin, which activates several genes. In the absence of a Wnt
uitin ligase complex, resulting in the degradation of ␤-cate-
nin (Latres et al., 1999). Activation of the receptor by its Wnt
ligands negatively regulates GSK-3␤, leading to the accumu-
lation of cytoplasmic ␤-catenin (Lee et al., 2003a).
Prostate cancer is one of the most frequently diagnosed
cancers and the second leading cause of cancer-related death
in men (Jemal et al., 2005). Dysfunction in the Wnt/␤-catenin
pathway is a frequent event during prostatic tumorigenesis
(
Barker and Clevers, 2000). For example, alterations in the
This work was supported by the Korea Science and Engineering Foundation APC gene, including somatic mutation and promoter hyper-
grant funded by the Korea government (Ministry of Science and Technology)
methylation, are frequently observed in prostate cancers, as
are mutations in the ␤-catenin gene (Chesire et al., 2000;
Gerstein et al., 2002). Indeed, the intracellular level of
(
R01-2006-000-10617-0) and Regional Innovation Center grants from Tradi-
tional and Bio-Medical Research Center, Daejeon University (RRC-04722,
2
006) by Industrial Technology Evaluation and Planning (ITEP).
Article, publication date, and citation information can be found at
␤-catenin is significantly elevated in more than 20% of ad-
http://molpharm.aspetjournals.org.
doi:10.1124/mol.107.040253.
vanced prostate tumors, 77% of prostatic lymph node metas-
ABBREVIATIONS: Fz, Frizzled; CRT, ␤-catenin response transcription; APC, adenomatous polyposis coli; GSK-3␤, glycogen synthase kinase-3␤;
CM, conditioned medium; ␤-TrCP, ␤-transducin repeat-containing protein; HEK, human embryonic kidney; hFz-1, human Frizzled-1; RT-PCR,
reverse transcription-polymerase chain reaction; DMSO, dimethyl sulfoxide; MG-132, N-benzyoloxycarbonyl (Z)-Leu-Leu-leucinal; TCF, T-cell
factor; AR, androgen receptor.
1599

1
600
Song et al.
tases, and 85% of prostatic bone metastases (Chesire and M. Davis (Hebrew University-Hadassah Medical School, Jerusalem,
Issac, 2002). ␤-Catenin is translocated into the nucleus, Israel). pCMV-RL and pSV-FL plasmids were purchased from
Promega.
where it complexes with T-cell factor (TCF)/lymphocyte en-
Screening for a Small-Molecule Inhibitor of Wnt/␤-Catenin
Signaling. The HEK293 reporter and control cell line was estab-
hancer factor family transcription factors to activate the ex-
pression of Wnt/␤-catenin-responsive genes, such as c-myc,
lished as described previously (Park et al., 2006). The cells were
cyclin D1, and metalloproteinase-7, which play important
inoculated into 96-well plates at 15,000 cells/well in duplicate and
roles in tumorigenesis and metastasis (He et al., 1998; Tetsu
grown for 24 h. Wnt3a CM was added, and then chemicals including
and McCormick, 1999; Takahashi et al., 2002). Thus, de-
coumarins, flavonoids, naphthoquinones, and terpenoids were added
crease of abnormally up-regulated intracellular ␤-catenin is
to the wells at a final concentration of 30 ␮g/ml. After 15 h, the plates
a potential therapeutic strategy for chemoprevention and were assayed for firefly luciferase activity and cell viability.
Western Blotting. The cytosolic fraction was prepared as de-
treatment of prostate cancer.
Decursin is a pyranocoumarin found in the roots of Angel- scribed previously (Dignam et al., 1983). Proteins were separated by
SDS-polyacrylamide gel electrophoresis in a 4 to 12% gradient gel
ica gigas Nakai, which has been traditionally used to treat
(
Invitrogen) and transferred to nitrocellulose membranes (Bio-Rad
anemia and other diseases (Chi and Kim, 1970). Decursin
has a cytotoxic effect on leukemia cells through a mechanism
involving protein kinase C and reactive oxygen species, and it
has antitumor activity in mice inoculated with sarcoma cells
Laboratories, Hercules, CA). The membranes were blocked with 5%
nonfat milk and probed with anti-␤-catenin (BD Transduction Lab-
oratories, Lexington, KY), anti-cyclin D1 (Santa Cruz Biotechnology,
Santa Cruz, CA), anti-myc (Santa Cruz Biotechnology), and anti-
actin antibodies (Cell Signaling Technology, Danvers, MA). The
membranes were then incubated with horseradish-peroxidase-conju-
(
Lee et al., 2003b; Kim et al., 2005). It has been reported that
decursin suppresses growth and promotes apoptosis in hu-
man prostate carcinoma cells (Yim et al., 2005; Jiang et al., gated anti-mouse IgG or anti-rabbit IgG (Santa Cruz Biotechnology)
006). Moreover, the (CH ) –CϭCH–COO– side chain of de- and visualized using the enhanced chemiluminescence system
2
3
2
cursin, which is replaced with an OH group in decursinol, (Santa Cruz Biotechnology).
RNA Extraction and Semiquantitative RT-PCR. Total RNA
seems to be necessary for its anticancer activity (Yim et al.,
005; Guo et al., 2007). In the present study, we identified
decursin as an inhibitor of the Wnt/␤-catenin pathway using
cell-based small-molecule screening. Decursin may inhibit
the growth of prostate cancer cells by promoting the degra-
dation of intracellular ␤-catenin.
was isolated with TRIzol reagent (Invitrogen) in accordance with the
manufacturer’s instructions. cDNA synthesis, reverse transcription,
and polymerase chain reaction were performed as described previ-
ously (Park et al., 2006). The amplified DNA was separated on 2%
agarose gels and stained with ethidium bromide.
Cell Viability Assay. Cells were inoculated into 96-well plates
and treated with decursin for 48 h. The cell viability from each
treated sample was measured in triplicate using CellTiter-Glo assay
kit (Promega) according to the manufacturer’s instructions.
2
Materials and Methods
Preparation of Decursin and Decursinol. (ϩ)-Decursinol was
prepared from the roots of A. gigas as described previously (Lee et al.,
Results
2
006). For the synthesis of decursin, decursinol was added to a
solution of 3,3-dimethylacrylic acid, dicyclohexylcarbodiimide, and
-dimethylaminopyridine with 350 ml of dry methylene chloride. The
Identification of Decursin as an Inhibitor of the
Wnt/␤-Catenin Pathway. To identify cell-permeable
small molecules capable of inhibiting the Wnt/␤-catenin
pathway, we used a cell-based screening system. HEK293
reporter cells that are stably transfected with TOPFlash, a
synthetic ␤-catenin/Tcf-dependent luciferase reporter, and
human Frizzled-1 expression plasmids were dispensed into a
4
reaction mixture was stirred at room temperature for 24 h and
filtered. The filtrate was evaporated under low pressure and purified
by flash chromatography (n-hexane/ethyl acetate ϭ 10:1 to 1:1) to
yield (ϩ)-decursin as a white powder. The structures of (ϩ)-decursi-
nol and (ϩ)-decursin were confirmed by comparing NMR and mass
spectrometry spectra, as reported previously (Lim et al., 2001).
Cell Culture, Transfection, and Luciferase Assay. HEK293, 96-well plate. After the addition of Wnt3a CM and each
PC-3, and Wnt3a-secreting L cells were obtained from American compound, we monitored TOPFlash reporter activity using a
Type Culture Collection (Manassas, VA) and maintained in Dulbec-
microplate reader (Fig. 1A). One of the compounds identified
co’s modified Eagle’s medium supplemented with 10% fetal bovine
serum, 120 ␮g/ml penicillin, and 200 ␮g/ml streptomycin. Wnt3a-
conditioned medium (Wnt3a CM) was prepared as described previ-
induced ␤-catenin response transcription (CRT); approxi-
ously (Gwak et al., 2006). In brief, Wnt3a-secreting L cells were
cultured in Dulbecco’s modified Eagle’s medium with 10% fetal bo-
from this screen was decursin (Fig. 1B). As shown in Fig. 1C,
decursin produced a dose-dependent decrease in Wnt3a CM-
mately 80% of CRT was blocked at a concentration of 200 ␮M
decursin. In contrast, activity of FOPFlash, a negative con-
trol reporter with mutated ␤-catenin/Tcf binding elements,
vine serum for 4 days. The medium was harvested and sterilized
using a 0.22-␮m filter. Fresh medium was added, the cells were
cultured for another 3 days, and the medium was collected and was not altered by incubation with decursin and Wnt3a CM
combined with the previous medium. Transfection was performed (Fig. 1C). Under these conditions, no cytotoxic effects of
with Lipofectamine 2000 (Invitrogen, Carlsbad, CA) according to the decursin were observed (data not shown). These results sug-
manufacturer’s instructions. The luciferase assay was performed
gest that decursin specifically inhibits the Wnt/␤-catenin
using a Dual Luciferase Assay kit (Promega, Madison, WI).
pathway.
Plasmid Constructs. Human Frizzled-1 cDNA was cloned as
Decursin Promoted Proteasome-Mediated ␤-Catenin
described previously (Cho et al., 2005). Reporter plasmids containing
Degradation. In the Wnt/␤-catenin pathway, CRT is largely
the cyclin D1 promoters were prepared by amplifying the promoter
controlled by the level of intracellular ␤-catenin, which reg-
ulates the expression of several target genes. To determine
whether decursin affects the intracellular level of ␤-catenin,
regions, which harbored TCF-4 response elements, by polymerase
chain reaction and insertion into pRL-null vectors, yielding pCy-
clinD1-RL. The pTOPFlash and pFOPFlash reporter plasmids were
obtained from Upstate Biotechnology (Lake Placid, NY). The domi- we used Western blot analysis with anti-␤-catenin antibody
nant-negative ␤-TrCP (⌬␤-TrCP) expression plasmid was a gift from to determine the amount of cytosolic ␤-catenin in decursin-

Decursin-Induced ␤-Catenin Degradation
1601
treated HEK293 reporter cells. As shown in Fig. 2A, incuba- protein degradation. Decursin induced a consistent decrease
tion with decursin resulted in down-regulation of the ␤-cate- in the level of ␤-catenin in HEK293 reporter cells (Fig. 2C);
nin that had accumulated in the presence of Wnt3a. We next however, the effect of decursin was nullified by the addition
examined whether decursin reduces the mRNA expression of of MG-132 (Fig. 2C). Taken together, these results indicate
␤-catenin in HEK293 reporter cells using semiquantitative that decursin inhibits the Wnt/␤-catenin pathway via protea-
RT-PCR. In contrast to the protein level of ␤-catenin, mRNA some-mediated degradation of ␤-catenin.
expression of ␤-catenin was unchanged by any of the concen-
␤-TrCP but Not GSK-3␤ Was Necessary for Decursin-
trations of decursin used, suggesting that decursin inhibits Mediated Degradation of ␤-Catenin. Because the phos-
Wnt/␤-catenin signaling by reducing ␤-catenin protein level phorylation of ␤-catenin by GSK-3␤ and its subsequent asso-
without affecting ␤-catenin mRNA expression (Fig. 2B). It ciation with ␤-TrCP leads to ␤-catenin degradation (Winston
was demonstrated previously that the intracellular level of et al., 1999), we examined whether decursin-mediated inhi-
␤-catenin is regulated by ubiquitin-dependent proteolysis bition of the Wnt/␤-catenin pathway requires GSK-3␤ activ-
(
Aberle et al., 1997). To explore whether the decursin-in- ity. To this end, HEK293 reporter cells were incubated with
duced down-regulation of ␤-catenin is mediated by the pro- LiCl, an inhibitor of GSK-3␤ (Klein and Melton, 1996), re-
teasome, we used MG-132 to inhibit proteasome-mediated sulting in increased CRT (Fig. 3A). It is interesting that
decursin suppressed LiCl-induced CRT (Fig. 3A). Further-
more, Western blot analysis showed that decursin reduced
the amount of ␤-catenin induced by LiCl (Fig. 3B), indicating
that decursin-mediated inhibition of the Wnt/␤-catenin
pathway is independent of GSK-3␤. We also tested whether
␤
␤
-TrCP is required for decursin-induced degradation of
-catenin. As shown in Fig. 3C, ectopic expression of a
dominant-negative form of ␤-TrCP (⌬␤-TrCP), which inter-
Fig. 2. Decursin promotes the degradation of ␤-catenin via a proteasome.
A, cytosolic proteins were prepared from HEK293 reporter cells treated
with either vehicle (DMSO) or indicated concentrations of decursin in the
presence of Wnt3a CM for 15 h and then subjected to Western blotting
with ␤-catenin antibody. B, semiquantitative RT-PCR for ␤-catenin, and
glyceraldehydes-3-phosphate dehydrogenase was performed with total
RNA prepared from HEK293 reporter cells either vehicle (DMSO) or
indicated concentrations of decursin in the presence of Wnt3a CM for
15 h. C, cytosolic proteins prepared from HEK293 reporter cells, which
were incubated with vehicle (DMSO) or decursin (100 ␮M) in the pres-
ence or absence of Wnt3a CM, exposed to MG-132 (20 ␮M) for 8 h, were
subjected to Western blotting with anti-␤-catenin antibody. In A and C, to
confirm equal loading, the blot was reprobed with anti-actin antibody.
Fig. 1. Identification of decursin as a small-molecule inhibitor of Wnt/␤-
catenin pathway. A, screening of compounds that inhibit Wnt/␤-catenin
pathway. Compounds modulating TOPFlash reporter activity were
screened using the HEK293 reporter cells. The controls were assayed in
the presence or absence of Wnt3a CM. B, chemical structure of decursin.
C, dose-dependent inhibition of CRT. HEK293 reporter and control cells
were incubated with indicated concentrations of decursin in the presence
of Wnt3a CM. After 15 h, luciferase activity was determined. TOPFlash
activity is reported as relative light unit (RLU) normalized to CellTiter-
Glo (Promega) activity. The results are the average of three experiments,
and the bars indicate standard deviations. ء, P Ͻ 0.05, and ءء, P Ͻ 0.01,
compared with the vehicle control group.

1
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Song et al.
acts with phosphorylated ␤-catenin but is unable to form a (Fig. 4A). In conjunction with this experiment, we evaluated
␤
-TrCP
SCF
ubiquitin ligase complex (Hart et al., 1999), abol- the level of cytosolic ␤-catenin in decursin-treated PC3 cells.
ished the decursin-mediated degradation of ␤-catenin. These Consistent with our results in HEK293 cells, Western blot
results suggest that decursin promotes the degradation of analysis showed that ␤-catenin was down-regulated in a
␤-catenin via a ␤-TrCP-dependent mechanism.
concentration-dependent manner (Fig. 4B). Furthermore,
Decursin Attenuated the Wnt/␤-Catenin Pathway in MG-132 abolished the decursin-induced down-regulation of
Prostate Cancer Cells. Because decursin was identified ␤-catenin in PC3 cells (Fig. 4C). These results indicate that
recently as a chemopreventive agent against prostate carci- decursin promotes ␤-catenin degradation in prostate cancer
noma cells (Yim et al., 2005; Jiang et al., 2006), we tested cells.
whether decursin also suppresses the Wnt/␤-catenin path-
Decursin Repressed the Expression of ␤-Catenin-De-
way in prostate cancer cells, which frequently show abnor- pendent Genes and Inhibited the Proliferation of
mally high levels of intracellular ␤-catenin (Barker and Clev- Prostate Cancer Cells. We next examined whether decur-
ers, 2000). Androgen-independent PC3 prostate cancer cells sin affects the expression of ␤-catenin-dependent genes. To
were transiently transfected with TOPFlash followed by in- this end, PC3 cells were transfected with a reporter construct
cubation with increasing amounts of decursin. As seen in the containing the cyclin D1 promoter, which contains a ␤-cate-
HEK293 reporter cells, decursin repressed CRT in PC3 cells nin/TCF-4-responsive region, and then incubated with differ-
ent concentrations of decursin. As shown in Fig. 5A, cyclin D1
promoter activity was repressed by decursin. We also mea-
sured the mRNA and protein expression of cyclin D1 in
decursin-treated PC3 cells. Consistent with our result for the
cyclin D1 promoter, a dose-dependent decrease in cyclin D1
Fig. 3. Decursin induces ␤-catenin degradation through a mechanism
independent of GSK-3␤ and dependent of ␤-TrCP. A, HEK293 reporter
cells were incubated with decursin in the presence of 20 mM LiCl. After
5 h, luciferase activity was determined. TOPFlash activity is reported as
Fig. 4. Decursin promotes the degradation of ␤-catenin in PC3 prostate
cancer cells. A, PC3 cells were cotransfected with TOPFlash and
pCMV-RL plasmids and incubated with decursin for 15 h. Luciferase
activities were measured 39 h after transfection. TOPFlash activity is
reported as relative light unit (RLU) normalized to Renilla reniformis
luciferase activity. Results are the average of three experiments, and the
bars indicate standard deviations. ءء, P Ͻ 0.01 compared with the vehicle
control group. B. cytosolic proteins were prepared from PC3 cells treated
with the vehicle (DMSO) or decursin for 15 h and then subjected to
Western blotting with ␤-catenin antibody. C, cytosolic proteins prepared
from PC3 cells were incubated with vehicle (DMSO) or decursin (100 ␮M),
exposed to MG-132 (20 ␮M) for 8 h, and then subjected to Western
blotting with anti-␤-catenin antibody. In B and C, to confirm equal
loading, the blot was reprobed with anti-actin antibody.
1
relative light unit (RLU) normalized to CellTiter-Glo (Promega) activity.
The results are the average of three experiments, and the bars indicate
standard deviations. ء, P Ͻ 0.05, and ءء, P Ͻ 0.01, compared with the
vehicle control group. B, cytosolic proteins were prepared from HEK293
reporter cells treated with either vehicle (DMSO) or increasing amounts
of decursin in the presence of 20 mM LiCl for 15 h and then subjected to
Western blotting with ␤-catenin antibody. C, HEK293 cells were trans-
fected with the ⌬␤-TrCP expression plasmid and then incubated with
either the vehicle (DMSO) or decursin (100 ␮M) in the presence of Wnt3a
CM for 15 h. Cytosolic proteins were subjected to Western blotting with
␤
-catenin antibody. In B and C, the blots were reprobed with anti-actin
antibody as a loading control.

Decursin-Induced ␤-Catenin Degradation
1603
mRNA and protein expression was observed in response to reported that decursinol has lower anticancer activity com-
decursin (Fig. 5, B and C). In addition, the expression of pared with decursin, suggesting that the missing side chain
c-myc, an established downstream target of ␤-catenin (He et may be responsible for the decursin-mediated growth inhibi-
al., 1998), was also significantly reduced in PC3 cells after
incubation with decursin (Fig. 5D). Recently, inhibition of the
Wnt/␤-catenin pathway by expression of Frzb/secreted Friz-
zled-related protein, a secreted Wnt antagonist, suppressed
the proliferation of androgen-dependent and -independent
prostate cancer cells (Zi et al., 2005; Horvath et al., 2007).
Moreover, cyclin D1, which forms a complex with cyclin-
dependent kinase 4/6, mediates growth factor-dependent G1
phase progression (Sherr, 1996). Given that decursin inhibits
the Wnt/␤-catenin pathway and cyclin D1 expression, we
examined whether decursin reduces PC3 cell growth. PC3
cells were incubated with varying concentrations of decursin,
and cell growth was monitored. As shown in Fig. 5E, decursin
efficiently suppressed PC3 cell growth in a concentration-
dependent manner.
tion of prostate cancer cells (Yim et al., 2005). To further
analyze the relationship between structure and function in
these compounds, we determined the effect of decursinol on
the Wnt/␤-catenin pathway. When HEK293 reporter cells
were incubated with decursinol, no significant inhibition of
Wnt3a-induced CRT was observed, in contrast to our results
with decursin (Fig. 6B). Consistent with this result, decursi-
nol did not decrease the amount of cytosolic ␤-catenin in
HEK293 reporter cells or in PC3 cells at any of the concen-
trations tested (Fig. 6, C and D), suggesting that decursinol
was unable to induce the degradation of ␤-catenin. As ex-
pected, decursinol did not repress the expression of cyclin D1
in PC3 cells (Fig. 6E), and it did not affect their growth at
concentrations of 50 and 100 ␮M, although it decreased their
growth by approximately 20% at a concentration of 200 ␮M
Decursinol Did Not Inhibit the Wnt/␤-Catenin Path-
(
Fig. 6F). Taken together, these results indicate that the
way. Decursinol is natural analog of decursin that lacks the
(
CH ) –CϭCH–COO– side chain (Fig. 6A). A recent study (CH
3
)
2
–CϭCH–COO– side chain of decursin is crucial for
3
2
Fig. 5. Decursin inhibits the expres-
sion of the ␤-catenin-dependent genes.
A, PC3 cells were cotransfected with
cyclin D1-RL and pSV40-FL and then
incubated with indicated amounts of
decursin for 15 h. Luciferase activities
were measured 39 h after transfection.
Cyclin D1 promoter activity is reported
as relative light unit (RLU) normalized
to firefly luciferase activity. The results
are the average of three experiments,
and the bars indicate standard devia-
tions. ء, P Ͻ 0.05 compared with the
vehicle control group. B, semiquantita-
tive RT-PCR for cyclin D1, and glycer-
aldehydes-3-phosphate dehydrogenase
was performed with total RNA pre-
pared from PC3 cells treated with the
vehicle (DMSO) or decursin for 15 h. C
and D, PC3 cells were incubated with
the vehicle (DMSO) or decursin for
1
5 h, and then cell extracts were pre-
pared for Western blotting with anti-
cyclin D1 (C) and anti-myc (D) antibod-
ies. To confirm equal loading, the blots
were reprobed with anti-actin antibody.
E, PC3 cells were incubated with in-
creasing amounts of decursin, and
cell viability was determined by CellTi-
ter-Glo assay (Promega). The results
shown are the average of three experi-
ments, and the bars indicate standard
deviations. ء, P Ͻ 0.05, and ءء, P Ͻ
0
.01, compared with the vehicle control
group.

1
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Song et al.
inhibition of the Wnt/␤-catenin pathway and the anticancer cellular ␤-catenin in the presence of Wnt3a and LiCl, which
activity of the compound in PC3 cells.
are well known GSK-3␤ inhibitors, suggesting that GSK3␤ is
not necessary for decursin-mediated ␤-catenin degradation.
In addition, overexpression of ⌬␤-TrCP abolished ␤-catenin
degradation by decursin, indicating that ␤-TrCP is required
for decursin-mediated ␤-catenin degradation. These data
suggest that decursin promotes the degradation of ␤-catenin
through a mechanism other than those described above. We
plan to investigate the mechanism of decursin-induced
Discussion
Aberrant accumulation of intracellular ␤-catenin is in-
volved in the progression of prostate cancer at an early stage,
during the formation of the primary lesion and at the ad-
vanced, hormone-refractory stage (Barker and Clevers,
2
000). Using a cell-based screen, we showed that decursin
␤-catenin degradation in the future.
suppresses the Wnt/␤-catenin pathway by promoting the
degradation of ␤-catenin. The level of intracellular ␤-catenin
is predominantly regulated by two pathways, a GSK3␤-de-
pendent pathway, and an Siah-dependent pathway. In the
GSK3␤-dependent pathway, the N-terminal residue of
Intracellular ␤-catenin promotes the development of pros-
tate cancer thorough the ␤-catenin/TCF and ␤-catenin/andro-
gen receptor (AR) pathways (Chesire and Issac, 2002). Acti-
vation of ␤-catenin/TCF pathway promotes proliferation and
suppresses apoptosis in prostate cancer cells (de la Taille et
al., 2003). In ␤-catenin/AR pathway, ␤-catenin interacts with
AR, resulting in the up-regulation of AR activity in a ligand-
dependent manner, which supports the growth of prostate
cancer cells (Mulholland et al., 2003). Recently, it was shown
␤-catenin is phosphorylated by a multiprotein complex com-
posed of APC, Axin, and GSK-3␤ (Hart et al., 1998), leading
to the degradation of ␤-catenin through a ubiquitin-depen-
dent mechanism (Aberle et al., 1997). In the Siah-dependent
pathway, Siah-1 interacts with the carboxyl terminus of
APC, which recruits the ubiquitination complex and pro- that decursin inhibits growth and promotes cell death in both
motes the degradation of ␤-catenin via a GSK-3␤- and androgen-dependent and -independent prostate cancer cells
␤
-TrCP-independent pathway (Liu et al., 2001). In this (Yim et al., 2005). Jiang et al. (2006) suggested that decursin
study, decursin was able to induce the degradation of intra- inhibits the proliferation of androgen-dependent LNCaP cells
Fig. 6. Decursinol does not inhibit
Wnt/␤-catenin pathway. A, chemical
structure of decursinol. B, HEK293
reporter cells were incubated with in-
dicated concentrations of decursinol
in the presence of Wnt3a CM. After
1
5 h, luciferase activity was deter-
mined. TOPFlash activity is reported
as relative light unit (RLU) normal-
ized to CellTiter-Glo (Promega) activ-
ity. The results are the average of
three experiments, and the bars in-
dicate standard deviations. ء, P Ͻ
0
.05 compared with the vehicle con-
trol group. C and D, cytosolic proteins
were prepared from HEK293 reporter
cells (C) or PC3 cells (D) treated with
either vehicle (DMSO) or indicated
concentrations of decursinol in the
presence of Wnt3a CM (C) for 15 h
and then subjected to Western blot-
ting with ␤-catenin antibody. E, cell
extracts were prepared from PC3 cell
treated with either vehicle (DMSO) or
indicated concentrations of decursinol
for Western blotting with anti-cyclin
D1 antibody. F, PC3 cells were incu-
bated with increasing amounts of
decursinol, and cell viability was de-
termined by CellTiter-Glo assay (Pro-
mega). The results shown are the av-
erage of three experiments, and the
bars indicate standard deviations. ء,
P Ͻ 0.05 compared with the vehicle
control group. In C, D, and E, the blots
were reprobed with anti-actin anti-
body to confirm equal loading.

Decursin-Induced ␤-Catenin Degradation
1605
via AR inactivation, including the inhibition of androgen-
stimulated nuclear translocation of AR and down-regulation
of AR protein expression. Here, we observed that decursin
suppressed CRT and promoted the degradation of intracellu-
lar ␤-catenin in androgen-independent PC3 cells. Moreover,
the expression of several targets of ␤-catenin, including cy-
clin D1 and c-myc, which play important roles in tumorigen-
esis and cell cycle progression (Utsunomiya et al., 2001), was
repressed by decursin in PC3 cells. This finding indicates
that decursin inhibits cellular proliferation by suppressing
the ␤-catenin/TCF pathway in androgen-independent cells
rather than by inactivating the ␤-catenin/AR pathway. Con-
sistent with this idea, recent studies have shown that inhi-
bition of the Wnt/␤-catenin pathway by expression of a Wnt
antagonist, such as Frzb/secreted Frizzled-related protein-3
and a dominant-negative version of LRP5, suppresses PC3
cell growth (Zi et al., 2005; Horvath et al., 2007).
de la Taille A, Rubin MA, Chen MW, Vacherot F, Burchardt M, Buttyan R, and
Chopin D (2003) ␤-catenin-related anomalies in apoptosis-resistant and hormone-
refractory prostate cancer cells. Clin Cancer Res 9:1801–1807.
Dignam JD, Lebovitz RM, and Roeder RG (1983) Accurate transcription initiation by
RNA polymerase II in a soluble extract from isolated mammalian nuclei. Nucleic
Acids Res 11:1475–1489.
Gerstein AV, Almeida TA, Zhao G, Chess E, Buhler K, Pienta K, Rubin MA, Vessella
R, and Papadopoulos N (2002) APC/CTNNB1 (␤-catenin) pathway alterations in
human prostate cancers. Genes Chromosomes Cancer 34:9–16.
Giles RH, van Es JH, and Clevers H (2003) Caught up in a Wnt storm: Wnt signaling
in cancer. Biochim Biophys Acta 1653:1–24.
Guo J, Jiang C, Wang Z, Lee HJ, Hu H, Malewicz B, Lee HJ, Lee JH, Baek NI, Jeong
JH, et al. (2007) A novel class of pyranocoumarin anti-androgen receptor signaling
compounds. Mol Cancer Ther 6:907–917.
Gwak J, Cho M, Gong SJ, Won J, Kim DE, Kim EY, Lee SS, Kim M, Kim TK, Shin
JG, et al. (2006) Protein-kinase-C-mediated beta-catenin phosphorylation nega-
tively regulates the Wnt/␤-catenin pathway. J Cell Sci 119:4702–4709.
Hart M, Concordet JP, Lassot I, Albert I, del los Santos R, Durand H, Perret C,
Rubinfeld B, Margottin F, Benarous R, et al. (1999) The F-box protein ␤-TrCP
associates with phosphorylated ␤-catenin and regulates its activity in the cell.
Curr Biol 9:207–210.
Hart MJ, del los Santos R, Albert IN, Rubinfeld B, and Polakis P (1998) Downregu-
lation of ␤-catenin by human Axin and its association with the APC tumor
suppressor, ␤-catenin and GSK3␤. Curr Biol 8:573–581.
He TC, Sparks AB, Rago C, Hermeking H, Zawel L, da Costa LT, Morin PJ,
Vogelstein B, and Kinzler KW (1998) Identification of c-MYC as a target of the
APC pathway. Science 281:1509–1512.
Decursinol, which contains a pyranocoumarin core with-
out the (CH ) –CϭCH–COO– side chain of decursin (Fig.
3
2
Horvath LG, Lelliott JE, Kench JG, Lee DS, Williams ED, Saunders DN, Grygiel JJ,
Sutherland RL, and Henshall SM (2007) Secreted Frizzled-related protein 4 in-
hibits proliferation and metastatic potential in prostate cancer. Prostate 67:1081–
6
A), is one of the most prevalent compounds in the roots of
Korean A. gigas (Yim et al., 2005). It was suggested pre-
viously that the side chain of decursin mediates its anti-
cancer activity against hormone-refractory DU145 pros-
tate cancer cells (Yim et al., 2005), and the side chain of
decursin was shown to confer anti-AR activity in LNCaP
prostate cancer cells (Guo et al., 2007). In this study,
decursinol lacked the properties of decursin; it had no
effect on Wnt3aCM-induced CRT, the intracellular ␤-cate-
nin level, or the expression of ␤-catenin-dependent genes
in HEK293 reporter and PC3 cells. It is interesting that
decursin had a strong antiproliferative effect on PC3 cells,
whereas decursinol did not (Figs. 5E and 6F). The different
effects of these compounds on the proliferation of PC3 cells
is in accord with their opposing effects on the Wnt/␤-
catenin pathway, suggesting that inhibition of the Wnt/␤-
catenin pathway is a central regulatory mechanism of
decursin-mediated inhibition of PC3 cell proliferation.
In conclusion, we investigated the anticancer effect of de-
cursin on androgen-independent prostate cancer cells using
cell-based screening. Decursin suppressed the Wnt/␤-catenin
pathway by promoting the degradation of ␤-catenin. We also
found that the (CH ) –CϭCH–COO– side chain of decursin is
1
090.
Jemal A, Murray T, Ward E, Samuels A, Tiwari RC, Ghafoor A, Feuer EJ, and Thun
MJ (2005) Cancer statistics, 2005. CA Cancer J Clin 55:10–30.
Jiang C, Lee HJ, Li GX, Guo J, Malewicz B, Zhao Y, Lee EO, Lee HJ, Lee JH, Kim
MS, et al. (2006) Potent antiandrogen and androgen receptor activities of an
Angelica gigas-containing herbal formulation: identification of decursin as a novel
and active compound with implications fro prevention and treatment of prostate
cancer. Cancer Res 66:453–463.
Kim HH, Ahn KS, Han H, Choung SY, Choi SY, and Kim IH (2005) Decursin and
PDBu: two PKC activators distinctively acting in the megakaryocytic differentia-
tion of K562 human erythroleukemia cells. Leuk Res 29:1407–1413.
Klein PS and Melton DA (1996) A molecular mechanism for the effect of lithium on
development. Proc Natl Acad Sci U S A 93:8455–8459.
2
ϩ
Kuhl M, Sheldahl LC, Park M, Miller JR, and Moon RT (2000) The Wnt/Ca
pathway: a new vertebrate Wnt signaling pathway takes shape. Trends Genet
6:279–283.
1
Latres E, Chiaur DS, and Pagano M (1999) The human F box protein ␤-Trcp
associates with the Cul1/Skp1 complex and regulates the stability of ␤-catenin.
Oncogene 18:849–854.
Lee E, Salic A, Kruger R, Heinrich R, and Kirschner MW (2003a) The roles of APC
and Axin derived from experimental and theoretical analysis of the Wnt path-
way [published erratum appears in PLoS Biol 2:E89, 2004]. PLoS Biol 1:E10.
Lee JH, Choi YS, Kim JH, Jeong HG, Kim DH, Yun MY, Kim EK, and Song GY
(
2006) A mass preparation method of (ϩ)-decursinol from the roots of Angelica
gigas. Yakhak Hoechi 50:338–344.
Lee S, Lee YS, Jung SH, Shin KH, Kim BK, and Kang SS (2003b) Antitumor activity
of decursinol angulated and decursin from Angelica gigas. Arch Pharmacol Res
26:727–730.
Lim JD, Kim IH, Kim HH, Ahn KS, and Han HG (2001) Enantioselective synthesis
of decursinol angelate and decursin. Tetrahedron Lett 43:4001.
Liu C, Li Y, Semenov M, Han C, Baeg GH, Tan Y, Zhang Z, Lin X, and He X (2002)
Control of ␤-catenin phosphorylation/degradation by a dual-kinase mechanism.
Cell 108:837–847.
3
2
crucial for its function, including inhibition of the Wnt/␤-
catenin pathway and cellular proliferation. Taken together,
our findings may facilitate the development of new antineo-
plastic therapeutics for prostate cancer.
Liu J, Stevens J, Rote CA, Yost HJ, Hu Y, Neufeld KL, White RL, and Matsunami N
(2001) Siah-1 mediates a novel ␤-catenin degradation pathway linking p53 to the
adenomatous polyposis coli protein. Mol Cell 7:927–936.
Mulholland DJ, Read JT, Rennie PS, Cox ME, and Nelson CC (2003) Functional
localization and competition between the androgen receptor and T-cell factor for
nuclear b-catenin: a means for inhibition of the Tcf signaling axis. Oncogene
Acknowledgments
22:5602–5613.
We thank M. Davis for dominant-negative ␤-TrCP expression
plasmid. We also thank D.-I. Kang for comments on the manuscripts.
Park S, Gwak J, Cho M, Song T, Won J, Kim DE, Shin JG, and Oh S (2006)
Hexachlorophene inhibits Wnt/␤-catenin pathway by promoting Siah-mediated
␤
-catenin degradation. Mol Pharmacol 70:960–966, 2006.
Polakis P (2000) Wnt signaling and cancer. Genes Dev 14:1837–1851.
Sherr CJ (1996) Cancer cell cycle. Science 274:1672–1677.
References
Takahashi M, Tsunoda T, Seiki M, Nakamura Y, and Furukawa Y (2002) Iden-
tification of membrane-type matrix metalloproteinase-1 as a target of the
␤-catenin/Tcf4 complex in human colorectal cancers. Oncogene 21:5861–
5867.
Tetsu O and McCormick F (1999) ␤-Catenin regulates expression of cyclin D1 in
colon carcinoma cells. Nature 398:422–426.
Utsunomiya T, Doki Y, Takemoto H, Shiozaki H, Yano M, Sekimoto M, Tamura S,
Yasuda T, Fujiwara Y, and Monden M (2001) Correlation of ␤-catenin and cyclin
D1 expression in colon cancers. Oncology 61:226–233.
Winklbauer R, Medina A, Swain RK, and Steinbeisser H (2001) Frizzled-7 sig-
nalling controls tissue separation during Xenopus gastrulation. Nature 413:
856–860.
Aberle H, Bauer A, Stappert J, Kispert A, and Kemler R (1997) ␤-catenin is a target
for the ubiquitin-proteasome pathway. EMBO J 16:3797–3804.
Barker N and Clevers H (2000) Catenins, Wnt signaling and cancer. Bioassays
22:961–965.
Chesire DR, Ewing CM, Gage WR, and Issac WB (2000) Detection and analysis of
-catenin mutation in prostate cancer. Prostate 45:323–334.
␤
Chesire DR and Issac WB (2002) Ligand-dependent inhibition of ␤-catenin/TCF
signaling by androgen receptor. Oncogene 21:8453–8469.
Chi HJ and Kim HS (1970) Studies on the components of Umbelliferae plants in
Korea: pharmacological study of decursin, decursinol and nodakenin. Korean
J Pharmacol 1:25–32.
Cho M, Gwak J, Park S, Won J, Kim DE, Yea SS, Cha IJ, Kim TK, Shin JG, and Oh
S (2005) Diclofenac attenuates Wnt/␤-catenin signaling in colon cancer cells by
activation of NF-␬B. FEBS Lett 579:4213–4218.
Winston JT, Strack P, Beer-Romero P, Chu CY, Elledge SJ, and Harper JW (1999)
␤
-TRCP
The SCF
-ubiquitin ligase complex associates specifically with phosphory-

1
606
Song et al.
lated destruction motifs in I␬B␣ and ␤-catenin and stimulates I␬B␣ ubiquitination
in vitro. Genes Dev 13:270–283.
Wodarz A and Nusse R (1998) Mechanisms of Wnt signaling in development. Annu
Rev Cell Dev Biol 14:59–88.
Expression of Frzb/secreted Frizzled-related protein 3, a secreted Wnt antagonist,
in human androgen-independent prostate cancer PC-3 cells suppresses tumor
growth and cellular invasiveness. Cancer Res 65:9762–9770.
Yim D, Singh RP, Agrwal C, Lee S, Chi H, and Agarwal R (2005) A novel anticancer
agent, decursin, induces G1 arrest and apoptosis in human prostate carcinoma
cells. Cancer Res 65:1035–1044.
Address correspondence to: Dr. Sangtaek Oh, PharmcoGenomics Research
Center, Inje University, Busan 614-735, Korea. E-mail: ohsa@inje.ac.kr
Zi X, Guo Y, Simoneau AR, Hope C, Xie J, Holcombe RF, and Hoang BH (2005)