A synthetic decursin analog with increased in vivo stability suppresses androgen receptor signaling in vitro and in vivo

Article abstract of DOI:10.1007/s10637-011-9738-x

Summary: Targeting androgen receptor (AR) signaling with agents distinct from current antagonist drugs remains a rational approach to the prevention and treatment of prostate cancer (PCa). Our previous studies have shown that decursin and isomer decursinol angelate (DA), isolated from the Korean medicinal herb Angelica gigas Nakai, interrupt AR signaling and possess anti-PCa activities in vitro. In the LNCaP PCa cell model, these pyranoccoumarin compounds exhibit properties distinct from currently used antagonists (e.g., Casodex). However, both are rapidly de-esterified to decursinol, a partial AR agonist. We report here that a synthetic decursin analog, decursinol phenylthiocarbamate (DPTC), has greater in vivo stability than the parent compounds. DPTC-decursinol conversion was undetectable in mice. Furthermore, in LNCaP cells, DPTC decreased prostate specific antigen (PSA) expression, down-regulated AR abundance and mRNA and inhibited AR nuclear translocation. The effect of DPTC on AR and PSA mRNA and protein abundance was also observed in VCaP cells expressing wild type AR. DPTC inhibited growth of both PCa cell lines through G₁ cell cycle arrest and apoptosis, as did decursin and DA. Furthermore, i.p. administration of DPTC for 3 weeks suppressed the expression of AR target genes probasin and Nkx3.1 in mouse prostate glands. Overall, our data suggest that DPTC represents a prototype lead compound for development of in vivo stable and active novel decursin analogs for the prevention or therapy of PCa.

Full text of DOI:10.1007/s10637-011-9738-x

Invest New Drugs (2012) 30:1820–1829
DOI 10.1007/s10637-011-9738-x
PRECLINICAL STUDIES
A synthetic decursin analog with increased in vivo stability
suppresses androgen receptor signaling in vitro and in vivo
Yong Zhang & Ahmad Ali Shaik & Chengguo Xing &
Yubo Chai & Li Li & Jinhui Zhang & Wei Zhang &
Sung-Hoon Kim & Junxuan Lü & Cheng Jiang
Received: 15 June 2011 /Accepted: 14 August 2011 /Published online: 26 August 2011
#
Springer Science+Business Media, LLC 2011
Summary Targeting androgen receptor (AR) signaling with
agents distinct from current antagonist drugs remains a
rational approach to the prevention and treatment of prostate
cancer (PCa). Our previous studies have shown that decursin
and isomer decursinol angelate (DA), isolated from the
Korean medicinal herb Angelica gigas Nakai, interrupt AR
signaling and possess anti-PCa activities in vitro. In the
LNCaP PCa cell model, these pyranoccoumarin compounds
exhibit properties distinct from currently used antagonists (e.g.,
Casodex). However, both are rapidly de-esterified to decursi-
nol, a partial AR agonist. We report here that a synthetic
decursin analog, decursinol phenylthiocarbamate (DPTC),
has greater in vivo stability than the parent compounds.
DPTC-decursinol conversion was undetectable in mice.
Furthermore, in LNCaP cells, DPTC decreased prostate
specific antigen (PSA) expression, down-regulated AR
abundance and mRNA and inhibited AR nuclear transloca-
tion. The effect of DPTC on AR and PSA mRNA and protein
abundance was also observed in VCaP cells expressing wild
type AR. DPTC inhibited growth of both PCa cell lines
through G1 cell cycle arrest and apoptosis, as did decursin
and DA. Furthermore, i.p. administration of DPTC for
3 weeks suppressed the expression of AR target genes
probasin and Nkx3.1 in mouse prostate glands. Overall, our
data suggest that DPTC represents a prototype lead com-
pound for development of in vivo stable and active novel
decursin analogs for the prevention or therapy of PCa.
Grant supports National Center for Complementary and Alternative
Medicine AT 005383, University of Minnesota Cancer Center Breast
Cancer Seed grant CON000000015215, Korea Science and
Engineering Foundation (KOSEF) grant No. 2011–0006220.
:
:
:
:
:
:
Y. Zhang A. A. Shaik Y. Chai L. Li J. Zhang S.-H. Kim
:
J. Lü C. Jiang
Hormel Institute, University of Minnesota,
801 16th Avenue NE,
Austin, MN 55912, USA
:
A. A. Shaik C. Xing
Department of Medicinal Chemistry, University of Minnesota,
Minneapolis, MN 55455, USA
.
.
Keywords Androgen receptor Decursin Stable
.
derivative Prostate cancer
W. Zhang
Southern Research Institute,
2000 Ninth Avenue South,
Birmingham, AL 35205, USA
Introduction
S.-H. Kim
Cancer Preventive Material Development Research Center,
College of Oriental Medicine, Kyunghee University,
Seoul 130-701, Republic of Korea
Prostate cancer (PCa) is the second leading cause of cancer
death in American men and is responsible for an estimated
30,000 deaths per year [1]. Androgen receptor (AR)
activation plays a crucial role in the genesis and progression
of PCa, and androgen deprivation therapies effectively
induce remission of the primary cancerous lesions [2–4].
However, relapse and progression to the incurable
castration-resistant prostate cancer (CRPCa) is virtually
inevitable [5]. Even CRPCa usually expresses AR protein
:
:
:
:
Y. Zhang L. Li J. Zhang J. Lü (*) C. Jiang (*)
Department of Biomedical Sciences, Texas Tech University Health
Sciences Center School of Pharmacy,
1300 S. Coulter St,
Amarillo, TX 79106, USA
e-mail: junxuan.lu@ttuhsc.edu
e-mail: Cheng.Jiang@ttuhsc.edu

Invest New Drugs (2012) 30:1820–1829
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A
and requires AR for survival [6, 7]. Abiraterone Acetate (a
CYP17 blocker inhibiting androgenic steroid synthesis) and
MDV-3100 (an inhibitor of AR ligand-binding activation)
have shown promise in recent clinical trials, inducing a
dramatic decline in serum prostate specific antigen (PSA)
and/or tumor regression, even in patients who have failed
current chemotherapy [8–10]. Overall, targeting AR signal-
ing with novel drugs distinct from current antagonist drugs
(e.g., Casodex/bicalutamide) remains an important and
rational approach to PCa prevention or therapy.
Decursin (D)
DecursinolAngelate
(DA)
Decursinol(DOH)
The dried root of Korean Angelica gigas Nakai (AGN)
has been used as a medicinal herb in Oriental countries for
millennia [11]. The major phytochemical components of
AGN include decursin and decursinol angelate (DA)
(Fig. 1a). These isomers share the same pyranocoumarin
core structure but are distinguished from each other by the
substituent group esterified to the 3′-position of the
core, i.e., senecioate and angelate for decursin and DA,
respectively (Fig. 1a).
Decursinol phenyl-
thiocarbamate
(DPTC)
B
Recent studies show that AGN-derived pyranocoumarins
inhibit growth and proliferation of various cancer cells in
culture, including prostate [12–17], bladder [18], breast [19],
colon [18], sarcoma [20] and leukemia [21]. Previous studies
from our group provide evidence that decursin and DA are
potent inhibitors of AR signaling in vitro, by a mechanism of
action distinct from the AR antagonist Casodex [12–14].
However, in vivo, both decursin and DA are rapidly de-
esterified to decursinol [16, 22], a compound having
antagonist activity at low micromolar concentrations but
partial agonist activity at increased levels in vitro [13]. This
observation suggests the hypothesis that the 3′-position
substituent determines the AR antagonist activity, and that
decursin analogs with non-hydrolyzable side chains may
exhibit increased stability and inhibitory potency in vivo.
In the present study, decursinol phenylthiocarbamate
(DPTC) was synthesized by coupling decursinol with
phenyl isothiocyanate (Fig. 1a). Our data provide experi-
mental evidence that DPTC was stable in vivo and exerted
inhibitory effect on AR signaling both in vitro and in vivo.
C
Fig. 1 a Molecular structures of decursin, decursinol angelate and
decursinol, and synthesis scheme of decursinol phenylthiocarbamate
(DPTC). Numbering follows Yim et al, [ref. 15]. b Plasma concen-
trations of DPTC and decursinol (DOH) after a single dose DPTC
(2 mg per mouse) via i.p. injection. c Plasma concentrations of
decursin/DA (D/DA) and decursinol (DOH) after a single dose of D/
DA mixture (4.8 mg per mouse) via i.p. injection. Each point
represents Mean SEM, n=3 mice
Materials and methods
thin layer chromatography (TLC) was performed on EM
Science silica gel 60 F254 (0.25 mm) (EM Industries Inc,
Gibbstown, NJ). Compounds were visualized by UV light
and further stained with p-anisaldehyde solutions followed
by heating. Flash column chromatography was performed
on Whatman Inc. silica gel (230–400 mesh). The 1H NMR,
13C NMR spectra were recorded on a BrukerAvance
Phenyl isothiocynate and sodium bis(trimethylsilyl)amide
(NaHMDS) were purchased from Sigma Aldrich (St. Louis,
MO). All other reagents and solvents were purchased from
Sigma Aldrich (St. Louis, MO) or Fischer Scientific
(Pittsburgh, PA). Unless otherwise stated, anhydrous solvents
were used without further purification or distillation. Decursin
and DA mixture were isolated from the ethanol extract of
Korean AGN root, following published procedures [23, 24].
All non-aqueous reactions were carried out under an
atmosphere of dry nitrogen in dried glassware. Analytical
1
400 MHz spectrometer. The chemical shifts for H NMR
were reported in ppm downfield to CDCl3 (7.26 ppm),
coupling constants were in Hz. ESI mode mass spectra
were recorded on a BrukerBiotofII mass spectrometer.
Optical rotations were recorded on a Rudolph Research

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Invest New Drugs (2012) 30:1820–1829
Autopol III polarimeter at 589 nm Na D-line. A Beckman
Coulter Gold liquid chromatographic system was utilized
for assessing the purity of synthesized compounds.
For cell culture studies, DPTC was dissolved in dimethyl
sulfoxide (DMSO) and stored at −20°C until used.
In vivo stability of DPTC The pharmacokinetic experiments
were approved by the Institutional Animal Care and Use
Committee of the University of Minnesota and carried out
at Hormel Institute animal facility. Adult C57BL/6 mice
were randomly divided into 6 groups of 3 animals. Each
animal received a single 2 mg dose of DPTC (~80 mg/kg)
delivered by i.p. injection. Another three mice received
only vehicle (DMSO: Ethanol: PEG400: Tween80: 5%
Glucose=1.5: 1.5: 6: 1: 20). At the indicated post-treatment
time, the blood was collected under isoflurane anesthesia
using heparinized capillary through a retro-orbital ap-
proach. The plasma concentrations of the indicated com-
pounds were measured using a Beckman System Gold liquid
chromatographic system [25]. In a separate experiment,
decursin and DA mixture (4.8 mg per mouse, ~192 mg/kg)
was given by i.p. injection and blood was collected at 3 early
time points based on our previous work [16] as a reference
for comparison.
Synthesis of decursinol phenylthiocarbamate (DPTC)
DPTC was synthesized in a two-step procedure (Fig. 1a).
Decursinol was first obtained through LiOH hydrolysis of
decursin and DA mixture. Briefly, anhydrous LiOH (6 eq)
was added to a solution of decursin and DA (1 eq) in
tetrahydrofuran (THF): H2O (4:1), and the mixture was
stirred overnight at room temperature. THF was removed
under reduced pressure. The pH of the aqueous layer was
adjusted to 2.0–3.0 with 1 N HCl and then extracted with
ethyl acetate (2 × 50 ml). The organic layers were
combined, washed with saturated NaCl solution and dried
over anhydrous MgSO4. The solvent was evaporated in
vaccuo and the residue was purified by flash column
chromatography on silica gel to yield decursinol (yield:
81%).
DPTC was obtained by coupling decursinol to phenyl
isothiocynate at the 3′-position of the pyranocoumarin core,
using NaHMDS. Decursinol (1 eq) was dissolved in dry
THF under a nitrogen atmosphere at −78°C. NaHMDS
(1.2 eq) was added drop-wise over a period of 15 min with
further stirring for another 30 min at −78°C. Phenyl
isothiocynate (1.1 eq) was added drop-wise to the reaction
mixture. After 1 h, the mixture was brought to room
temperature and stirred for an additional 6 h. The progress
of the reaction was followed by TLC. At completion, the
reaction was quenched with saturated aqueous ammonium
chloride and extracted with ethyl acetate. The organic layers
were combined, washed with saturated NaCl solution and
dried over anhydrous MgSO4. The solvent was evaporated
in vacuuo and the residue was purified by flash column
chromatography on silica gel with a DPTC yield of 66%.
The purity was assessed by HPLC, using a 5 μM
CLIPEUS C 18 column (250mmX4.6 mm, Higgins),
Mobile phase A: Water, B: Acetonitrile. Elution was by a
linear gradient from 35% B for 1 min, 35%B to 95%B in
25 min, and then kept at 95%B for 10 min. The Flow rate
was 0.45 ml/min and detection wavelength 277 nm. The
purity was great than 99%.
Cell culture Human androgen-responsive prostate cancer
LNCaP (mutant AR, T877A) and VCaP (wild type AR)
cells were purchased from American Type Culture Collec-
tion (ATCC, Manassas, VA). Except when indicated
otherwise, the cells were grown and treated in the complete
ATCC-recommended growth medium supplemented with
10% fetal bovine serum without antibiotics under the
standard 37°C and 5% CO2 humidified environment.
ELISA measurement of secreted PSA After exposure to
indicated treatments, the medium of LNCaP cells was
collected and the floating cells were removed by centrifu-
gation. The secreted PSA in the growth medium was
quantified using a MAGIWEL PSA ELISA system (United
Biotech, Mountain View, CA) as previously described [12,
26].
RNA isolation and real-time PCR Total RNA was isolated
from PCa cells or mouse prostate tissue using RNeasy Mini
kit (Qiagen, Valencia, CA) according to the user manual.
Total RNA (2 μg) from each sample was reverse tran-
scribed using SuperScript™ II RT Kit (Invitrogen, Carls-
bad, CA). Real-time PCR was performed on an Applied
Biosystems 7500 machine (Applied Biosystems, Carlsbad,
CA) using FastStart Universal SYBR Green Master (Rox)
system (Roche Diagnostics GmbH, Mannheim, Germany).
Human AR primers: forward 5′-CAG GAG GAA GGA
GAG GCT TC-3′, reverse 5′-AGC AAG GCT GCA AAG
GAG TC-3′. Human PSA primers: forward 5′-CCC ACT
GCA TCA GGA ACA AA-3′, reverse 5′-GAG CGG GTG
TGG GAA GCT-3′. Mouse probasin primers: forward 5′-
The chemical properties and molecular structure of
DPTC were: TLC (EtOAc : Hexane=1:2) Rf=0.26; pale
yellow solid; [α]D +47.7 (c=1.0, CHCl3); 1H NMR
25
(400 MHz, CDCl3): δ 8.37 (1H, brs), 7.56 (1H, d,
J=9.6 Hz), 7.31 (1H, s), 7.17–6.90 (5H, m), 6.81 (1H, s),
6.23 (1H, d, J=9.6 Hz), 5.77 (1H, t, J=3.98 Hz), 3.27 (1H,
d, J=17.2 Hz ), 3.13 (1H, d, J=17.2 Hz), 1.39 (6H, s); 13C
NMR (100 MHz, CDCl3): δ 187.2, 161.3, 156.5, 154.3,
143.2, 136.5, 129.1, 129.0, 125.6, 123.3, 121.0, 115.3,
113.5, 113.0, 104.6, 78.6, 27.6, 24.7, 24.3; ESI-MS
(positive): m/z 382 (M+1)+.

Invest New Drugs (2012) 30:1820–1829
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ATC ATC CTC CTG CTC ACA CTG CAT G-3′, reverse 5′-
ACA GTT GTC CGT GTC CAT GAT ACG C-3′. Mouse
Nkx3.1 primers: forward 5′-ACT GAA CCC GAG TCT
GAT GCA C-3′, reverse 5′-TAC AGG TAG GGG TAG TAG
GGA TAG C-3′ [27, 28]. β-actin was used for template
normalization. The PCR condition was started as 1 cycle of
2 min at 50°C and 10 min at 95°C, then 40 cycles of 15 s at
95°C and 1 min at 60°C, and then 1 cycle of 15 s at 95°C,
1 min at 60°C, 15 s at 95°C and 15 s at 60°C.
medium change. The adherent cells were fixed using 1%
glutaraldehyde, and the cell number was estimated after
staining with crystal violet (0.02%).
In vivo activity The in vivo anti-AR signaling property of
DPTC was probed in male adult C57BL/6 mice (~18 weeks
of age). Experimental animals received an i.p. injection
once daily for 5 days of DPTC (2 mg per mouse, ~80 mg/
kg); control mice were treated with vehicle alone (DMSO:
Ethanol: PEG400: Tween80: 5% Glucose=1.5: 1.5: 6: 1:
20) (once daily, 5 days per week). The general health status
of the mice was monitored daily. Approximately 1 h after
the last treatment, blood was collected under isoflurane
anesthesia before euthanizing the animals by CO2 inhala-
tion. The whole prostate of each mouse was collected and
total RNA extracted. Real-time PCR was performed to
detect the mRNA levels of mouse AR target genes Nkx3.1
and probasin [27, 28]. β-actin was used for template
normalization.
Immuno-blotting Whole cell lysate or subcellular fractions
were prepared for immuno-blotting as previously described
[12]. Anti-PSA was obtained from DAKO (Glostrup,
Denmark). Anti-AR and anti-α-tubulin were from Santa
Cruz Biotechnology Inc (Santa Cruz, CA). Anti-poly ADP-
ribose polymerase (PARP) and anti-cleaved PARP were
obtained from Cell Signaling Technology (Beverly, MA).
Measurement of AR degradation LNCaP cells were treated
with cycloheximide (an inhibitor of protein synthesis,
obtained from Sigma-Aldrich, St Louis, MO) to block
new protein synthesis with or without DPTC exposure for
3, 6 or 12 h, and then the cellular AR abundance was
quantified by immuno-blotting as previously described [13,
29].
Statistical analyses Student’s two-tailed t-test was used to
determine the statistical significance between specific
groups.
Molecular modeling In silico docking was performed using
the Schrödinger Suite 2009 (Schrödinger, LLC) [31] based
on the cyproterone acetate (CPA) bound complex crystal
structure of T877A AR ligand binding domain (AR-LBD).
The Induced Fit Docking (IFD) protocol [32], which takes
into consideration the ligand-induced receptor conforma-
tional change, was used for all docking studies. Residues
within 5Å from ligands were allowed to be flexible. The
docking results were scored using the Extra-Precision mode
of Glide version 5.0 (Schrödinger, LLC) [33]. The IFD
protocol and parameters were validated by docking CPA to
the T877A AR-LBD which reproduced the protein-ligand
binding mode in its complex crystal structure. The same
protocol and parameters were then used to study the
docking of pyranocoumarin compounds.
AR nuclear translocation Evaluation of AR nuclear trans-
location was carried out as previously described [12, 13,
29]. In brief, nuclear AR was depleted from LNCaP cells
by growing them in phenol-red free RPMI-1640 growth
medium supplemented with 5% charcoal stripped serum for
48 h. After pre-treatment with DPTC for 1 h, the cells were
stimulated with mibolerone (a dihydrotestosterone [DHT]
analog, kindly provided by Dr. Charles Young, Mayo
Clinic, Rochester, MN) for another 2 h. The nuclear and
cytosolic fractions of the cell lysate were separated using
the NE-PER Nuclear and Cytoplasmic Extraction Reagents
Kit (Pierce, Rockford, IL). The AR abundance in each
fraction was quantified by immuno-blotting, using PARP
and α-tubulin as markers of nuclear and cytosolic fractions,
respectively.
Results
Cell cycle distribution PCa cells were exposed to DPTC for
24 h before labeling those cells actively engaged in DNA
synthesis using deoxybromouridine BrdU (final concentra-
tion 1 μM, 30 min exposure). The cells were collected for
cell cycle distribution analysis as previously described [30].
Synthesis and in vivo stability of decursinol
phenylthiocarbamte (DPTC)
Synthesis of DPTC from decursinol was achieved by
coupling phenyl isothiocynate to the 3′-position of the
pyranocoumarin core of decursinol using NaHMDS. The
thiocarbamate functionality was chosen based on the well-
established knowledge of the relative stability of thiocarba-
mate>amide>ester, and was expected to decrease the rate
Colony growth assay Colony growth assay was used to
evaluate the growth response of LNCaP cells to DPTC as
previously described [12, 29]. In brief, LNCaP cells were
seeded on 6-well plates (6 × 103 cells per well) and exposed
to a single DPTC treatment for 13 days without growth

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Invest New Drugs (2012) 30:1820–1829
of hydrolysis. The phenyl functional group was incorporat-
ed because of the easy access to such a starting material and
the fact that the position for the phenyl group has been
found to be able to accommodate various alkyl and aryl
functional groups. The product was purified using flash
column chromatography on silica gel. The molecular
LNCaP cells, showing that DPTC preserved the AR
degradation activity of decursin or DA [13].
While DPTC also suppressed PSA secretion in VCaP
cells, it did so only at a much higher concentration than
for LNCaP cells (Fig. 2a, right). The cellular PSA and
AR abundance (Fig. 2b, right) and mRNA (Fig. 2c, right)
in VCaP cells responded to DPTC in a similar manner as
in LNCaP cells.
1
structure of DPTC was confirmed by H, 13C NMR and
ESI-MS. Its purity as assessed using HPLC was over 99%.
In previous pharmacokinetic studies, decursin and
decursinol angelate (DA) were rapidly hydrolyzed to
decursinol in rats and mice, as indicated by the rapid
increase of plasma decursinol [16, 22]. The in vivo stability
of DPTC was evaluated in a similar pharmacokinetic
experiment. As shown in Fig. 1b, after a single i.p.
injection, the DPTC plasma peak concentration was
reached at 30 min and declined with t1/2~1 h. The rapid
clearance suggests this compound may be conjugated (e.g.,
to glutathione) following uptake and excreted quickly. The
plasma concentration of decursinol was below limit of
detection at all 6 time points. When decursin/DA mixture
(4.8 mg per mouse, i.p.) was administered, the rapid
disappearance of decursin/DA and sharp rise in plasma
decursinol (Fig. 1c) confirmed the unstable nature of these
compounds [16, 22]. Taken together, the data support a
substantial improvement of in vivo stability of DPTC
compared with decursin and DA.
DPTC inhibited AR nuclear translocation in LNCaP cells
The transcriptional activity of the AR-ligand complex
depends on translocation from the cytoplasm to the
nucleus. The effect of DPTC on AR nuclear transloca-
tion was evaluated in androgen-deprived LNCaP cells
stimulated with mibolerone, a dihydrotestosterone
[DHT] analog. As shown in Fig. 2e, AR resided largely
in the cytoplasm during androgen deprivation (lane 1 vs.
2), but quickly translocated into the nucleus after
mibolerone stimulation (lanes 1 and 2 vs. 3 and 4). DPTC
rapidly (2 h) and significantly inhibited this ligand
activated nuclear translocation (lanes 3 and 4 vs. 7 and
8). These data suggest that, in LNCaP cells, DPTC inhibits
AR signaling not only through down-regulation of AR
abundance, but also through inhibition of AR nuclear
translocation.
DPTC down-regulated cellular AR and PSA abundance
in LNCaP and VCaP cell models
DPTC induced cell cycle arrests and apoptosis
The cell cycle and death impact of DPTC was tested in
LNCaP and VCaP cells by exposing them to DPTC for
24 h and evaluating both the cell cycle distribution and
apoptosis. In both cell lines, 25 μM DPTC significantly
increased G1 arrest, and reduced S phase cells (Fig. 3a); at
50 μM DPTC increased accumulation of cells at the G2/M
phases (Fig. 3a). Caspase-mediated apoptosis was elevat-
ed, as indicated by increased cleaved PARP, a well-known
marker of caspase-mediated execution (Fig. 3b). The
BrdU incorporation rate after 24 h of DPTC exposure
indicated a virtual shut-off of DNA replication regardless
of the cell cycle arrest status (Fig. 3a). Colony growth
assay of LNCaP cells showed that DPTC (>2.5 μM)
induced growth inhibition in a concentration-dependent
manner (Fig. 3c). Taken together, the data suggest that
DPTC suppresses PCa cell growth through induction of
cell cycle arrests and apoptosis, as do decursin and DA
[12, 13].
To evaluate the AR inhibitory activity of DPTC, PSA
expression was measured in two cell lines that express PSA
in response to AR agonists. LNCaP cells express a mutant
but functional AR (T877A) while VCaP cells express wild
type AR. As shown in Fig. 2a, DPTC exposure decreased
secreted PSA in LNCaP in a concentration-dependent
manner with IC50 of ~6 μM. In comparison, the IC50 for
decursin or DA under same treatment regimen was ~2 μM
[12, 13]. As with decursin or DA treatment of LNCaP,
DPTC decreased cellular PSA protein (Fig. 2b, left) and
mRNA abundances (Fig. 2c, left) in a concentration- and
time-dependent manner.
As shown in Fig. 2b, treatment with DPTC decreased
cellular AR protein abundance (Fig. 2b), as well as AR
mRNA steady state abundance (Fig. 2c, left). The latter
observation suggests an additional level of AR regulation
for DPTC as compared to decursin or DA, which do not
affect AR mRNA [12, 13].
Our earlier work has shown that decursin or DA
accelerates AR protein degradation through the proteoso-
mal pathway in LNCaP cells [13]. When new protein
synthesis was blocked by cycloheximide, DPTC shortened
the half-time of AR protein (Fig. 2d, lanes 2–4 vs. 5–7) in
DPTC down-regulated AR target genes in mouse prostates
The effect of chronic DPTC administration to inhibit AR
signaling in vivo was explored in adult mice. Animals
received daily i.p. injections of either DPTC (2 mg per

Invest New Drugs (2012) 30:1820–1829
Fig. 2 Inhibition of AR signal-
1825
A
LNCaP
*
VCaP
*
ing by DPTC in LNCaP and
VCaP PCa cells. a ELISA de-
tection of secreted PSA protein
(S-PSA) in the growth medium
of LNCaP cells and VCaP cells
after 48 h of DPTC exposure.
Each column represents Mean
SEM, n=3. b Immuno-blotting
detection of cellular PSA and
AR protein in LNCaP (left pan-
el) and VCaP (right panel) cells
after DPTC exposure for indi-
*
**
***
***
cated duration. c Real-time RT-
PCR detection of steady state
B
LNCaP
VCaP
levels of PSA and AR mRNA in
LNCaP (left panel) and VCaP
(right panel) cells after 24 h of
DPTC exposure. Each column
represents Mean SEM, n=3. d
Immuno-blotting detection of
steady state level of AR protein
in LNCaP cells after new protein
synthesis was blocked by cy-
cloheximide (CHX). e Effect of
DPTC on ligand-binding acti-
VCaP
C
vated AR nuclear translocation
LNCaP
in LNCaP cells. PARP and α-
tubulin were markers of the
nuclear and cytosolic fractions,
respectively. C: cytosolic frac-
tion, N: nuclear fraction. Mib:
mibolerone. Statistical signifi-
cance from the control, *P<
0.05, **P<0.01, ***P<0.001
*
*
**
**
**
**
**
**
D
E
1
2
3
4
5
6 7
mouse) or vehicle for 3 weeks (n=4 mice per group). There
was no significant effect of DPTC on body weight
(Fig. 4a). The plasma concentration of DPTC was approx-
imate 2.1 μM (Fig. 4b), and there was no detectable
decursinol in plasma (Fig. 4b). Compared to the vehicle
control group, DPTC significantly decreased the mRNA
abundance of AR target genes Nkx3.1 [27] and probasin
[28] (Fig. 4c). As an androgen-responsive organ, the
prostate of the DPTC-treated group weighed ~80% of that
of the vehicle-treated group, albeit not statistically signif-
icant (Fig. 5d). Taken together, the data suggested a
probable inhibition of AR signaling by DPTC at the target
gene level in vivo.
Molecular modeling of the potential interaction
between DPTC and T877A AR-LBD
Molecular docking was performed to gain insight into
the interactions between DPTC and the AR ligand
binding domain (AR-LBD). As the T877A mutation
expressed by LNCaP cells is a super-functional AR, it
was chosen for the docking simulation. Both DPTC
(Fig. 5a–b) and the natural pyranocoumarin compounds
(Fig. 5c–e) were able to dock in the binding pocket of
T877A AR-LBD. The estimated binding free energies
(docking scores) were −12.4 (DPTC), -11.3 (decursin), -11.5
(DA) and −9.5 (decursinol) kcal/mol. The relatively higher

1826
Invest New Drugs (2012) 30:1820–1829
Fig. 3 Effect of DPTC on cell
cycle, apoptosis and colony
growth of PCa cells. a Cell
cycle distribution and BrdU in-
corporation rate of LNCaP (left
panel) and VCaP (right panel)
cells exposed to DPTC for 24 h.
Each column represents Mean
SEM, n=3. b Immuno-blotting
detection of cleaved PARP
(cPARP) in LNCaP (upper pan-
el) and VCaP (lower panel) cells
after DPTC exposure for indi-
cated duration. c Representative
image of colony growth of
LNCaP
A
B
VCaP
*
**
*
*
*
*
**
**
*
**
**
**
C
LNCaP
LNCaP cells exposed to DPTC
treatment for 13 days and esti-
mation of the adherent cells by
crystal violet staining. Each
point represents Mean SEM,
n=3; Statistical significance
from the control, *P<0.05,
**P<0.01, ***P<0.001
***
***
***
***
predicted binding affinities of decursin and DA as compared
to decursinol were consistent with our previous report that
decursin was a more potent inhibitor of DHT binding to AR
T877A in LNCaP cells [13]. The energetically more
favorable docking score of DPTC as compared to decursin
and DA (−12.4 vs. −11.3 and −11.5 kcal/mol) would suggest
a higher binding affinity than its natural counterparts,
assuming free diffusion of these compounds.
Detailed structure analyses (Fig. 5b–e) suggested that the
interactions with T877A AR-LBD was very similar for
DPTC, decursin and DA. The ketone oxygen at 2-position
of these molecules forms a hydrogen bond with ARG752,
located in the interior of the binding pocket. At the other
end of the molecule, the various hydrophobic substituents
interact with the hydrophobic residues near the entrance of
the binding pocket, including PHE697, PHE876, LEU880
and LEU701. In contrast, decursinol appears to occupy a
similar region as the heterocyclic ring fragment of the
bulkier pyranocoumarins, but with the entire molecule
flipped over so that the hydroxyl group approaches the
inner side of the binding pocket, forming a hydrogen bond
with the side chain of GLN711. This flip-over is likely
caused by the expected unfavorable interaction between the
hydroxyl group and the near-entrance hydrophobic envi-
Fig. 4 Down-regulation of AR-
A
B
target genes by DPTC in mouse
prostates after 3 weeks of daily
i.p. injection at a dosage of 2 mg
per mouse. a Body weight (BW)
of each group (n=4). b Plasma
concentrations of DPTC and
decursinol (DOH). §: DPTC
below the detection limit; #:
DOH below the detection limit.
c Real-time RT-PCR detection of
#
#
&
steady state levels of probasin
and Nkx3.1 mRNA in mouse
D
C
prostates. d Prostate weight
(PW) of each group. Veh and
DPTC: vehicle- and DPTC-
treated groups. Statistical signif-
icance from the control,
**
*P<0.05, **P<0.01
*

Invest New Drugs (2012) 30:1820–1829
1827
A
B
C
Decursin: -11.3 kcal/mol
DPTC: -12.4 kcal/mol:
D
E
Decursinol angelate: -11.5 kcal/mol
Decursinol: -9.5 kcal/mol
Fig. 5 Structural representation of molecular docking. a Structural
model of T877A AR-LBD and DPTC complex. T877A AR-LBD was
shown in carton and colored by secondary structures; residues near
DPTC were shown in wires; DPTC was represented in solid sticks. b–
e, Close up view of protein ligand interactions between T877A AR-
LBD and DPTC (b), decursin (c), decursinol angelate (d) and
decursinol (e). Ligands were represented in solid sticks, key AR
residues near ligands were shown in wires, and hydrogen bonds were
represented in dashed yellow lines. Docking scores were shown on
each image

1828
Invest New Drugs (2012) 30:1820–1829
ronment. Such unique binding mode of decursinol might, in
part, explain its distinct actions on AR signaling as
compared to bulkier natural pyranocoumarins [13].
unique flipped-over docking mode of decursinol (Fig. 5e)
forces hydrogen bonding with the GLN711 side chain.
These differences might, in part, explain the AR antagonist
activities of DPTC, decrusin and DA and the partial agonist
activity of decursinol.
Discussion
It is noteworthy that daily administration of DPTC for
3 weeks significantly suppressed AR target gene expression
in mouse prostate (Fig. 4c) without affecting mouse body
weight (Fig. 4a). These data suggest that DPTC does not act
at the putative membrane-bound anabolic AR signaling axis
[35]. Since human and mouse share the same AR-LBD, this
selective antagonism might also occur in humans. The in
vitro data from human VCaP prostate cancer cells which
possess wild type AR lend general support to this
prediction (Fig. 2 and 3).
In spite of its advantages with regard to receptor
specificity and resistance to hydrolysis, the limited
water-solubility of DPTC is a challenge for efficient
delivery. Precipitation from cell culture media might
reduce its cellular uptake substantially. More impor-
tantly, particulate deposits visible on major organs in the
mouse abdominal cavity after 3 weeks of i.p. treatment
suggest that the drug also precipitates from peritoneal
fluid. The inferior hydrophilicity of DPTC compared to
decursin might provide a plausible explanation for its
decreased inhibitory potency in cell culture models with
respect to PSA and AR readouts and overall cell growth.
Anticipating that more hydrophilic stable decursin analogs
might achieve greater potency in vivo, we are carrying out
synthesis of additional decursinol thiocarbamate analogs
and efficacy evaluations.
Our previous studies show that decursin and its isomer DA
are potent inhibitors of AR signaling in vitro in the LNCaP
cell model and their mechanisms of action are distinct from
the AR antagonist Casodex [12, 13]. Casodex stimulates
AR translocation and stabilizes AR in LNCaP model [34],
whereas decursin and DA block AR nuclear translocation
and decrease AR abundance at least in part through
triggering proteosomal degradation [13]. Casodex acts as
a partial AR agonist under androgen deprivation in LNCaP
model [13, 34], but decursin and DA lack such agonist
effect [13].
The naturally occurring side chain substituent of decru-
sin and DA confers persistent anti-AR signaling and growth
inhibitory actions in vitro[13]. However, in vivo both
compounds are rapidly metabolized to decursinol through
hydrolysis of the labile ester bond linking the side chains to
decursinol [16, 22], greatly reducing their potential clinical
utility. We predict that a non-hydrolyzable side chain would
stabilize the molecule. In the present study, a decursin
analog DPTC has been synthesized, having a phenyl-
thiocarbamate group at the 3′-position (Fig. 1a). Pharma-
cokinetic studies in mice show that DPTC is more resistant
to hydrolysis than either decursin or DA, and that DPTC is
not converted back to decursinol (Fig. 1b and 4b).
In LNCaP cells, DPTC retained the inhibitory activity
and mechanistic attributes of decursin or DA against AR
signaling [12, 13], albeit with reduced potency. As did
decursin and DA, DPTC suppressed AR-dependent tran-
scription, down-regulated AR protein abundance and
retarded AR nuclear translocation (Fig. 2). Whereas
decursin induces G1 arrest for androgen independent
DU145 cells [15], DPTC inhibited androgen-responsive
LNCaP and VCaP PCa cell growth through inducing G1
cell cycle arrest at non-apoptotic exposure and G2/M arrest
and caspase-mediated apoptosis (Fig. 3), same as did
decursin and DA in LNCaP cells [13]. Furthermore, DPTC
also reduced AR mRNA abundance (Fig. 2c), a property
not shared with decursin or DA [12, 13], adding a unique
aspect to its molecular action.
Molecular modeling (Fig. 5) offers insight into the
structural interaction between the pyranocoumarins and
T877A AR-LBD. DPTC, decursin and DA share the same
binding pose (Fig. 5b–d), where the 2-position ketone
interacts with ARG752 deep in the binding pocket while
the hydrophobic side chains interact with hydrophobic
amino acids near the entrance of the pocket. In contrast, the
In summary, the present study suggests that stabilizing
the side chain structure of decursin-like compounds is a
promising and feasible approach to improve their in vivo
anti-AR efficacy. DPTC represents an important prototype
lead compound for the development of novel AR antago-
nists for the prevention or therapy of PCa.
Acknowledgement We acknowledge the assistance of Ellen Kroc
and animal facility staff and Todd Schuster of Shared Instruments Core
facility for help with flow cytometric analyses. We thank Dr. Margret
Weis for extensive copy editing of the manuscript during revision.
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