Potent Antiandrogen and Androgen Receptor Activities of an

Potent Antiandrogen and Androgen Receptor Activities of an

Angelica gigas–Containing Herbal Formulation: Identification

of Decursin as a Novel and Active Compound with Implications

for Prevention and Treatment of Prostate Cancer

Cheng Jiang,1Hyo-Jeong Lee,

1,2Guang-xun Li,

1Junming Guo,

1Barbara Malewicz,

1Yan Zhao,

1

Eun-Ok Lee,2Hyo-Jung Lee,

2Jae-Ho Lee,

2Min-Seok Kim,

2Sung-Hoon Kim,

2and Junxuan Lu

1

1Hormel Institute, University of Minnesota, Austin, Minnesota and 2Graduate School of East-West Medical Science,Kyunghee University, Seoul, South Korea

Abstract

Androgen and androgen receptor (AR)–mediated signalingare crucial for the development of prostate cancer. Identi-fication of novel and naturally occurring phytochemicalsthat target androgen and AR signaling from Orientalmedicinal herbs holds exciting promises for the chemo-prevention of this disease. In this article, we report thediscovery of strong and long-lasting antiandrogen and ARactivities of the ethanol extract of a herbal formula (termedKMKKT) containing Korean Angelica gigas Nakai (AGN) rootand nine other Oriental herbs in the androgen-dependentLNCaP human prostate cancer cell model. The functionalbiomarkers evaluated included a suppression of the expres-sion of prostate-specific antigen (PSA) mRNA and protein(IC50, f7 Mg/mL, 48-hour exposure) and an inhibition ofandrogen-induced cell proliferation through G1 arrest and ofthe ability of androgen to suppress neuroendocrine differ-entiation at exposure concentrations that did not causeapoptosis. Through activity-guided fractionation, we identi-fied decursin from AGN as a novel antiandrogen and ARcompound with an IC50 of f0.4 Mg/mL (1.3 Mmol/L, 48-hourexposure) for suppressing PSA expression. Decursin alsorecapitulated the neuroendocrine differentiation inductionand G1 arrest actions of the AGN and KMKKT extracts.Mechanistically, decursin in its neat form or as a componentof AGN or KMKKT extracts inhibited androgen-stimulatedAR translocation to the nucleus and down-regulated ARprotein abundance without affecting the AR mRNA level. Thenovel antiandrogen and AR activities of decursin anddecursin-containing herbal extracts have significant implica-tions for the chemoprevention and treatment of prostatecancer and other androgen-dependent diseases. (Cancer Res2006; 66(1): 453-63)

Introduction

Prostate cancer is second only to lung cancer as the cause ofcancer deaths in American men, responsible for an estimated30,000 deaths per year (1). Cytotoxic chemotherapy and radiother-apy provide no survival benefit to patients with hormone-refractoryprostate cancer, which usually arises after hormonal ablationtherapies (2–4). Presently, only some taxane drugs have shownlimited efficacy for advanced prostate cancer (5). All of thesetreatments cause significant side effects. Nontoxic alternatives areneeded to decrease the risk and burden of prostate cancer.Chemoprevention has become recognized as a plausible and

cost-effective approach to reducing cancer morbidity and mortalityby inhibiting precancerous events before the occurrence of clinicaldisease (6, 7). Androgen and androgen receptor (AR)–mediatedsignaling are crucial for the development and function of thenormal prostate (2) as well as for prostate cancer (2, 3). Theimportance of androgen in prostate cancer is supported bythe observations that prostate cancer rarely occurs in eunuchs orin men with deficiency in 5a-reductases, the enzymes that converttestosterone to its active metabolite 5a-dihydrotestosterone (2, 3).Targeting androgen and AR signaling represents a rational strategyfor the chemoprevention of prostate cancer (6, 7). As a proof ofconcept, a clinical trial with finasteride (Proscar), which inhibits5a-reductase II within the prostate gland, had shown a significantreduction of total prostate cancer incidence (8). However, prostatecancer that developed in subjects in the intervention group seemedto be more advanced in tumor stages than those from the placebogroup, raising doubt about the overall survival benefit of this single-target approach. Novel agents that target multiple aspects ofandrogen and AR signaling will be more desirable.Oriental herbal medicine has been used since ancient times to

treat malignancies. Systematic characterization of active phyto-chemicals in medicinal herbs and their mechanisms of action areimportant for providing the rationale for their efficacy and fortransforming herbal practices into evidence-based medicine. Incollaborative work aimed at developing safe and efficacious Orientalherbal formulas for prostate cancer chemoprevention, our labora-tories focused on herbs and their phytochemicals that targetandrogen and AR signaling as well as cell cycle arrest and apoptosis.With respect to biomarkers of androgen and AR signaling, prostate-specific antigen (PSA) is a gene tightly regulated by androgen innormal prostate and some prostate cancer cells (9). A member of thekallikrein family (KLN3), PSA is a serine protease with highlyprostate-specific expression and is elevated in blood circulation ofpatients with prostate cancer. Circulating PSA is widely used

Note: M-S. Kim is currently at the Department of Molecular Biology, University ofWisconsin, Madison, WI 53706. The University of Minnesota is an equal opportunityeducator and employer. J. Lu and S-H. Kim are senior corresponding authors.

Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/).

Requests for reprints: Junxuan Lu, Hormel Institute, University of Minnesota, 80116th Avenue Northeast, Austin, MN 55912. Phone: 507-437-9680; Fax: 507-437-9606;E-mail: [email protected]. or Sung-Hoon Kim, Graduate School of East-West MedicalScience, Kyunghee University, 1 Seochunri Kiheungeup, Yongin 449-701, South Korea.Phone: 82-31-201-2179; Fax: 82-31-205-1074; E-mail: [email protected]

I2006 American Association for Cancer Research.doi:10.1158/0008-5472.CAN-05-1865

www.aacrjournals.org 453 Cancer Res 2006; 66: (1). January 1, 2006

Research Article

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clinically as a marker for prostate cancer screening and isparticularly useful as an indicator of prostate cancer response totherapy and recurrence (9). The LNCaP human prostate cancer cellsare perhaps the best-studied in vitro model for androgen and ARsignaling in prostate cancer. They possess a high-affinity mutant ARand produce high levels of PSA, which is extremely responsive toandrogen stimulation (10, 11). In addition to PSA expression,another known outcome of androgen deprivation or blockage of ARsignaling in these cells is an induction of neurite-like projectionsthat have been termed neuroendocrine differentiation (12–15).Androgen signaling represses thesemorphologic manifestations andthe associated molecular markers, such as neuron-specific enolase(NSE; ref. 15). Therefore, we chose this cell line as the primary celltargets for screening novel antiandrogen and AR agents using PSAand neuroendocrine differentiation as key functional biomarkers.We report here strong antiandrogen and AR activities of the

ethanol extract of an anticancer formula (KMKKT) containingKorean Angelica gigas Nakai (Umbelliferae family; AGN) root andnine other herbs in LNCaP cells. We describe, for the first time,the identification of decursin from AGN as a novel antiandrogenand AR agent. Decursin was first isolated from Angelica decursiva(Fr. et Sav.) in Japan in 1966 and later from AGN (16, 17). Decursinhas been reported to induce cytotoxic activity of leukemia celllines in vitro (17, 18) and to be active against sarcoma growth inan animal model (19). For prostate cancer cells, a recent articlehas shown G1 cell cycle arrest and apoptosis induced by decursinin LNCaP, DU145, and PC-3 cell lines (20). The antiandrogen andAR activities described in the present article, together with thereported antiproliferative and apoptotic activities of decursin inprostate cancer cells, have significant implications for thechemoprevention and treatment of prostate cancer with decursinand decursin-containing herbs and formulas.

Materials and Methods

KMKKT. This herbal formula was originally designed to promote vitalenergy, to remove blood stasis, and to decrease inflammation for treating

lung cancer. The 10 ingredients and their proportion (w/w) were as follows:

Benincasa hispida , China, 17.2%; Bletilla striata , China, 8.6%; Tulipa edulis ,Korea, 8.6%; Panax ginseng , Korea, 8.6%; Phaseolus angularis , Korea, 17.2%;

Zanthoxylum piperitum , Korea, 6.9%; Patrinia villosa , China, 8.6%; Astragalus

membranaceus , Korea, 8.6%; Asini gelatinum , Korea, 8.6%; and AGN, Korea,

6.9%. The herbal ingredients were obtained from Oriental Medical Hospitalof Kyunghee University (Seoul, Korea) and kindly authenticated by Dr. Nam-

In Baek (Department of Oriental Herbal Materials, Kyunghee University). In

studies to be reported elsewhere, the ethanol extract of this formula given

by i.p. injection every other day decreased the growth of human PC-3prostate cancer tumor xenograft in athymic nude mice by 68% with a dose

of 100 mg/kg and decreased the growth of transplantable mouse Lewis lung

carcinoma in syngeneic mice by 86%, without any adverse effect on bodyweight of the mice.3

Extraction of KMKKT and constituent herbs. The ethanol extract wasprepared as follows: The dried and pulverized medicinal herbs were mixed

together and each 350-g batch was soaked with ethanol (2 liters� 3 changes)at room temperature for 7 days. The extract was filtered through filter paper

(pore size, 3 Am), evaporated (rotary evaporator, model NE-1, Japan), and

lyophilized ( freeze dryer, Lioalfa-6, Telstar, Terrassa, Spain) to produce 12.9 g

of powder (yield, 3.7%). The individual herbs were extracted by the sameprocedure for each 200-g batch with 1 liter ethanol � 3 changes. The yield

(w/w) was as follows: B. hispida , 1.76%; B. striata , 5.8%; T. edulis , 1.2%;

P. ginseng , 2.15%; P. angularis , 1.69%; Z. piperitum , 7.15%; P. villosa , 10.6%;A. membranaceus , 4.12%; A. gelatinum , 0.05%; and AGN, 6.48%.

Fractionation of AGN extract. AGN powder (1.2 kg) was obtained from

Kyung-Dong Pharmaceutical Co. (Seoul, Korea) and was extracted thrice

with ethanol. After filtration, concentration, and freeze drying as described

above, the ethanol extract (253.6 g) was obtained (yield, f21.1%). The

ethanol extract was reconstituted in distilled water and partitioned with an

equal volume of n-hexane. The resulting aqueous phase was partitioned in

succession with an equal volume of methylene chloride, ethyl acetate, and

butanol, respectively. The n-hexane, methylene chloride, ethyl acetate, and

butanol fractions and the residual liquid were concentrated and lyophilized.

The yields for the various fractions as percentage of input AGN ethanol

extract were as follows: n-hexane fraction (2.94 g, 1.16%), methylene

chloride fraction (35.2 g, 13.8%), ethyl acetate fraction (16.0 g, 6.3%), butanol

fraction (12.2 g, 4.8%), and residue (17.0 g, 6.7%).

Purified and synthetic decursin. Decursin was extracted and purified

by methods reported in ref. 19. The purity was determined to be f98.6%. A

crystalline sample (100%) of synthetic decursin (18) was kindly provided byProf. Hogyu Han (Department of Chemistry, Korea University, Seoul, Korea).

Assay for decursin. Decursin standard and extract/fraction samples

were spotted on Silica Gel 60 thin-layer plates (20 � 20 cm; Merck,

Darmstadt, Germany). After development in hexane/ethyl acetate (1:2 v/v),10% sulfuric acid was sprayed on the TLC plates to char carbon-containing

compounds. A high-performance liquid chromatography (HPLC) method

was also developed to measure decursin contents in herbal extracts(Supplementary Fig. S1).

Cell culture and treatments. LNCaP cells were obtained from the

American Type Culture Collection (Manassas, VA) and grown in RPMI 1640

supplemented with 10% fetal bovine serum (Atlanta Biologicals, Lawrence-

ville, GA) and 2 mmol/L L-glutamine, 10 mmol/L HEPES, 1 mmol/L sodium

pyruvate, and 45 g/L glucose without antibiotics. When cells were 50% to

60% confluent (usually 48 hours after plating), the medium was changed

and treatment with ethanol extracts of KMKKT, its constituent herbs, or

purified decursin was started. To standardize the condition of agent

exposure, cells were cultured in a volume-to-surface area ratio of 0.2 mL/cm2

(15 mL for a T75 flask and 5 mL for a T25 flask).In experiments where androgen stimulation was required, cells were

seeded in phenol red–free medium containing 5% charcoal-stripped serum

(CCS; Atlanta Biologicals) to decrease background signaling. Androgen

stimulation was provided by a nonmetabolizable analogue mibolerone,which was a kind gift from Dr. Charles Young (Mayo Clinic, Rochester, MN).

Bicalutamide, also known as Casodex, was purchased from LKT Labs

(St. Paul, MN) and used as a positive antiandrogen agent for comparison

with decursin in key experiments.Cell growth and cell death/apoptosis assays. The effect of herbal

extracts on cell number was estimated by either quantitating cell protein

yield by Lowry method or by the mitochondrial metabolism-based 2,3-bis[2-methoxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5-carboxanilide, inner

salt or 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfo-

phenyl)-2H-tetrazolium, inner salt (MTS) colorimetric methods in 96-well

plate format (21). Cell cycle analyses were carried out with propidiumiodide staining according to Krishan’s protocol (22) and flow cytometry

using a FACSCalibur flow cytometer (Becton Dickinson, San Jose, CA).

Cell death was detected by either an ELISA kit purchased from Roche

Diagnostics Corp. (Indianapolis, IN; ref. 23) or by an immunoblot analysisof the caspase-mediated cleavage of poly(ADP-ribose) polymerase (PARP)

as described previously (24).

ELISA for PSA protein in conditioned medium and cellular extract.An assay kit from United Biotech, Inc. (Mountain View, CA) was used for

measurement of PSA in both conditioned medium (secreted) and cell lysate

(cellular) as described previously (25).

Reverse transcription-PCR for mRNA of PSA, AR, NSE, andglyceraldehyde-3-phosphate dehydrogenase. After treatment for the

desired duration, LNCaP cells were used for RNA extraction by RNeasy kit

(Qiagen, Valencia, CA). Reverse transcription was done with 3 to 4 Ag total

RNA and oligo(dT) primers using SuperScript II RT (Life Technologies,Foster City, CA). PCR was carried out using Qiagen HotStarTaq Master Mix3 H-Je. Lee, S-H. Kim, and J. Lu, in preparation.

Cancer Research

Cancer Res 2006; 66: (1). January 1, 2006 454 www.aacrjournals.org



kit under optimized conditions for detecting differences in transcriptabundance. Oligonucleotide primers were synthesized by Sigma-Genosys

(The Woodlands, TX) as follows: (a) PSA gene (710 bp), 22 cycles, forward

5V-GATGACTCCAGCCACGACCT-3V and reverse 5V-CACAGACACCCCAT-CCTATC-3V, annealing temperature 57jC; (b) AR gene (590 bp), 32 cycles,forward 5V-ATGGAAGTGCAGTTAGGG-3V and reverse 5V-CAGGATGTCTTT-AAGGTCAGC-3V, annealing temperature 57jC; (c) NSE gene (662 bp;

Genbank X51956), 40 cycles, forward 5V-GTTCTGAACGTCTGGCTAAATAC-3V and reverse 5V-CATTGAGTTATGGGGAAATGA-3V, annealing temperature60jC; and (d) housekeeping gene glyceraldehyde-3-phosphate dehydrogenase

(230 bp), 25 cycles, forward 5V-TCAAGAAGGTGGTGAAGCAG-3Vand reverse

5V-CTTACTCCTTGGAGGCCATG-3V, annealing temperature 57jC.Immunoblotting of PSA, AR, A-tubulin, and cleaved and total PARP.

Cell lysate preparation and immunoblotting were as described previously

(24, 25). Antibody for PSA was purchased from DAKO (Glostrup, Denmark),

and an antibody for ARwas purchased from BD PharMingen (San Diego, CA).In experiments assessing effects of decursin or herbal extracts on AR

nuclear translocation, the cells were harvested and separated into nuclear

and cytosolic fractions using NE-PER Nuclear and Cytoplasmic Extraction

Reagents kit (Pierce, Rockford, IL) and the specificity of separation wasconfirmed by immunoblotting for total PARP (antibody from Cell Signaling

Technologies, Beverly, MA) and a-tubulin (antibody from Santa Cruz

Laboratories, Santa Cruz, CA) as the respective nuclear and cytosol markers.

PSA promoter-luciferase assay. LNCaP cells were cotransfected witha luciferase reporter plasmid driven by a 6-kb PSA promoter and a

cytomegalovirus (CMV)-h-galactosidase (h-gal) vector (generous gifts of

Dr. Charles Young) and incubated in serum-free, phenol red–free mediumfor 24 hours to reduce AR signaling as described previously (25). The cells

were treated with different levels of decursin plus androgen (0.1 nmol/L

mibolerone) for 24 hours in 5% CSS medium. Cell extracts were prepared for

luciferase and h-gal assays. Luciferase activities were normalized to h-galactivities to correct for differences in transfection efficiency.

Neuroendocrine differentiation. LNCaP cells were seeded into T25

flasks to f20% confluence with phenol red–free medium containing 5%

CSS unless noted otherwise in some experiments in which 5% whole serumwas used. Treatment with KMKKT, AGN, or decursin was initiated at

seeding time in most experiments. Androgen stimulation with mibolerone

was started 2 days after seeding. The overall extent of neuroendocrinedifferentiation for each flask was estimated by examining at least five

random fields under �100 magnification. Then, representative morphologic

changes were recorded with a CCD camera.

Replications and statistical evaluations. Each result was replicated inat least two independent experiments, and many were done multiple times.

When appropriate and necessary, ANOVAs and two-tailed t tests were used

to test statistical significance of differences from untreated control.

Results

Effects of KMKKT on cell growth and apoptosis. As shown inFig. 1A (left), KMKKT treatment for 48 hours in complete mediumcontaining 10% whole serum inhibited LNCaP cell growth in aconcentration-dependent manner in the range of 20 to 100 Ag/mL.At z200 Ag/mL and longer exposure (72 hours), KMKKT inducedapoptotic DNA fragmentation (Fig. 1A, right) and increased PARPcleavage (data not shown). The growth suppression effect wasaccompanied by G1 arrest (Table 1). Therefore, KMKKT caused G1

arrest at low concentration levels and apoptosis at z200 Ag/mLconcentrations.KMKKT exerts a sustained inhibition of PSA expression. We

next determined the effect of nonapoptotic concentrations ofKMKKT on PSA expression and secretion after 48-hour exposure.As shown in Fig. 1B (left), KMKKT decreased cellular PSA proteinlevel and PSA secreted into the conditioned medium in aconcentration-dependent manner. The IC50 was estimated to bef7 Ag/mL based on three independent experiments. In time

course experiments, exposure to 50 Ag/mL KMKKT for 6 hours didnot decrease cellular PSA level and by 12 hours decreased cellularPSA by half (Fig. 1B, right). By 72 hours, the cellular PSA expressionwas almost completely blocked (Fig. 1B). The secreted PSAfollowed the same kinetics of suppression as the cellular PSA.To determine whether the effect of KMKKT occurred at the

transcription level, we examined the steady-state level of the PSAmRNA over a course of exposure of 48 to 96 hours (Fig. 1C, left) and inthe acute duration of 9 to 24 hours (Fig. 1C, right). We includedmethylseleninic acid as a positive treatment that we had shownearlier to decrease PSA mRNA and AR mRNA (25) to validate thereverse transcription-PCR (RT-PCR) detection methodology (Fig. 1C,left). The RT-PCR assay detected a significant reduction of PSAmRNA abundance at 48 and 96 hours of KMKKT treatment (Fig. 1C),in excellent agreement with the ELISA data in Fig. 1B . KMKKTtreatment decreased the PSAmRNA level starting at 9 hours (Fig. 1C,right) and in a concentration-dependent manner when examinedat 24 hours of exposure (Fig. 1C, right).The AR mRNA was not decreased during KMKKT treatment

from 9 to 96 hours (Fig. 1C). Because AR function is necessary forPSA transcription, we analyzed by immunoblot whether KMKKTdecreased AR protein abundance and found that treatment for24 hours significantly decreased AR protein level (Fig. 1D). Theseresults indicate that the suppressing action of KMKKT on PSAprotein level was largely mediated by decreasing PSA mRNAtranscript abundance, which was accompanied by decreased ARprotein level without changing the level of AR mRNA.KMKKT inhibits androgen-stimulated AR nuclear translo-

cation. To determine whether KMKKT affects AR nucleartranslocation, we grew LNCaP cells in phenol red–free mediumsupplemented with 5% CSS for 2 days to decrease basal signaling.The cells were pretreated with increasing concentrations ofKMKKT extract for 1 hour and were stimulated with mibolerone(1 nmol/L) for 2 hours in the continued presence of KMKKTextract. Nuclear and cytosolic fractions were prepared forimmunoblot analyses. As shown in Fig. 1E , PARP and a-tubulindetection confirmed the specificity of nuclear and cytosolpreparations. Mibolerone stimulation converted a predominantlycytosolic distribution pattern for AR under androgen-deprivedstate (Fig. 1E, lanes 1 and 2) to one that was mostly nuclearlocalized (lanes 3 and 4). KMKKT extract decreased nuclear ARlevel in a concentration-dependent manner (compare lanes 5 and 7with lane 3). These results indicate that KMKKT treatment rapidlyinhibited androgen-stimulated AR nuclear translocation in addi-tion to the suppression of AR protein abundance.KMKKT induces expression of NSE and morphologic

features of neuroendocrine differentiation. It has been knownthat deprivation of androgen or a blockage of AR signaling bybicalutamide leads to neuroendocrine differentiation in LNCaP cellsand to the induction of molecular markers, such as NSE (12, 15). Wetherefore examined the mRNA level of NSE and found thisneuroendocrine differentiation marker gene significantly increasedby as little as 20 Ag/mL KMKKT at 24 hours of exposure in thecomplete medium (Fig. 1C, right, lanes14-16 versus lane 13) and theNSE induction was sustained through 96 hours (Fig. 1C, left, lane 6versus lane 5, lane 8 versus lane 7). The NSE expression data provideda molecular clue for the likely induction of neuroendocrine differen-tiation by KMKKT under the appropriate cell seeding conditions.To detect the morphologic features of neuroendocrine differen-

tiation, we treated sparsely plated LNCaP cells in 5% whole serum–supplemented medium with a single exposure of KMKKT. By 7

Antiandrogen Activities of Decursin and Angelica gigas




days after KMKKT treatment, f70% cells displayed ‘‘neurite’’morphology (Fig. 2A, b), whereas only f10% of the control cellsshowed such a feature (Fig. 2A, a). As a positive control (15), LNCaPcells in phenol red–free medium supplemented with 5% CSS formedneurites in f60% of the cells (Fig. 2A, c), which was effectivelyreversed by mibolerone (Fig. 2A, d). Under the experimentalconditions described in Fig. 2A (a and b), a single treatment withKMKKT induced and sustained neuroendocrine differentiationwithout cell loss for 4 weeks when the experiment was terminated(data not shown).To further establish the efficacy of KMKKT to specifically

block androgen actions in LNCaP cells in phenol red–freemedium with CSS, we titrated the mibolerone concentrationequivalent of KMKKT using neuroendocrine differentiation andPSA expression as biomarkers (Fig. 2B). In the absence ofKMKKT treatment, as little as 0.1 nmol/L mibolerone signifi-cantly increased LNCaP cell proliferation as evident from

photographs (Fig. 2B, top, DMSO) and protein yield (Fig. 2C ,control) and also increased PSA secretion (Fig. 2D, control).Maximal growth and PSA responses were obtained with0.2 nmol/L mibolerone. In the concentration range of 0.1 to 1nmol/L, mibolerone significantly decreased the proportion ofcells with neurite projections (Fig. 2B, top). In the presence of 50Ag/mL KMKKT, z0.5 nmol/L mibolerone was needed to reverseneuroendocrine differentiation (Fig. 2B, middle) and to partiallyrestore cell proliferation (Fig. 2C). With 100 Ag/mL KMKKT, z1nmol/L concentration of mibolerone was required to overrideneuroendocrine differentiation (Fig. 2B, bottom) and to partiallyrestore cell proliferation (Fig. 2C). Both concentrations ofKMKKT greatly suppressed the androgen-stimulated PSA secre-tion into the medium (Fig. 2D). Together, these data supportedstrong and lasting inhibitory activities on androgen-stimulatedcell proliferation and PSA expression and on the androgensuppression of neuroendocrine differentiation by KMKKT.

Figure 1. A, effect of KMKKT extract on LNCaP cellgrowth in the complete medium as estimated by proteinyield (48 hours; left ) and cell apoptosis as estimated byCell Death ELISA kit quantifying oligonucleosomal DNAfragmentation (72 hours; right ). For cell growth assay,exponentially growing cells (f50% confluent) in T25 flaskswere exposed to KMKKT in the complete medium for48 hours. Points (left), mean of three independentexperiments; bars, SD. For cell death assay, the cellswere treated the same but harvested after 72 hours oftreatment. Both adherent and detached cells werecombined for the death assay. B, concentration-dependent inhibition of cellular PSA protein expressionand secretion (left ) and time course of inhibition of cellularPSA level (right ) by KMKKT extract. Cellular PSA(by ELISA) was normalized to total protein. Secreted PSA(by ELISA) was measured in the conditioned medium. Inconcentration response experiments, LNCaP cells weregrown in T25 flasks and exposed to DMSO solvent(control) or KMKKT in the complete medium for48 hours. Points, mean of three independent experiments;bars, SD. IC50 was f7 Ag/mL. In time course experiment,exponentially growing LNCaP cells in T25 flasks werecarefully washed once with serum-free medium and werereplaced fresh medium (as time 0) containing either DMSOsolvent (control) or KMKKT (50 Ag/mL). C, effects ofKMKKT on the steady-state level of mRNA transcripts ofPSA, AR, and NSE between exposure duration of 48 to 96hours (left) and 9 to 24 hours (right ) detected by RT-PCR.NSE mRNA was detected as a molecular marker forneuroendocrine differentiation. Methylseleninic acid(MSeA ) was used as a positive control treatment tovalidate the detection method for PSA and AR mRNA byRT-PCR. D, immunoblot detection of effects of KMKKTextract on cellular PSA protein and AR protein levels inLNCaP cells after 24-hour exposure in the completemedium. E, inhibitory effect of KMKKT on AR nucleartranslocation stimulated by a nonmetabolizable androgenanalogue mibolerone. LNCaP cells were grown in phenolred–free medium supplemented with 5% CSS for 2 days.The cells were pretreated with increasing concentrationsof KMKKT extract for 1 hour and were stimulated with1 nmol/L mibolerone for 2 hours in the continued presenceof KMKKT extract (total exposure time of 3 hours).Nuclear (N ) and cytosolic (C ) fractions were prepared forimmunoblot analyses. PARP and a-tubulin were detectedas markers of nuclear and cytosol proteins, respectively.

Cancer Research




A. gigas extract contains antiandrogen agents. To identifythe active herb(s), we prepared the ethanol extract of each of the10 herbs and evaluated their effects individually on PSA expression.Mixing all 10 extracts in the proportion used for the formulation ofKMKKT reconstituted the PSA-suppressing efficacy (Fig. 3A , column3, Mix). When each extract was tested at 20 Ag/mL, AGN extractcompletely suppressed cellular PSA (Fig. 3A, column 17), whereas A.gelatinum came second potent among the 10 herbs (Fig. 3A, column12). The IC50 for AGN extract was estimated to be f1 Ag/mL(Fig. 3A) and that for A. gelatinum was f15 Ag/mL (data notshown). Due to its low extraction yield of f0.05% by ethanol, theactual contribution of A. gelatinum to the antiandrogen and ARactivities of KMKKTcould be much less than this differential in IC50

values. Herbs 1-8 (Fig. 3A, columns 4-11) had negligible effect (within30% of control) at the concentration of 20 Ag/mL.Decursin is a novel antiandrogen and AR compound. Next,

we fractionated AGN extract to identify the active compound(s).Because decursin is a known major component of Korean AGNwith cytotoxic activity in leukemia cells (17, 18), we focused onthis phytochemical (structure shown in Fig. 3D) as a likelycandidate. As shown in Fig. 3B , TLC analyses showed thepresence of a small amount of decursin in KMKKT (lane 2). HPLCanalysis estimated this content to be f1.7% (SupplementaryFig. S1). The decursin content was enriched in the methylenechloride (lane 5) and ethyl acetate (lane 6) fractions incomparison with the starting AGN extract (lane 3), whereasits content was decreased in the butanol fraction (lane 7). Then-hexane, methylene chloride, and ethyl acetate fractions weremore potent than the AGN extract for suppressing cellular PSA,whereas the butanol fraction was less efficacious (Fig. 3C).Similarly, these fractions showed the same order of potency insuppressing LNCaP cell viability/growth after 24-hour exposure(Fig. 3C). The decursin content in general correlated andpredicted the potency of each solvent fraction in suppressingPSA and cell growth (Fig. 3C).

Indeed, exposure of LNCaP cells to purified decursin (purity,f98.6%) for 48 hours in complete medium decreased cellular andsecreted PSA with IC50 of f0.4 Ag/mL (1.3 Amol/L; Fig. 4A). Syn-thetic crystalline decursin (18) generously provided by Prof. HogyuHan produced the same potency and pattern of inhibition (data notshown). Exposure for 24 hours to decursin (1.6, 3.3, and6.6 Ag/mL) and AGN extract (5, 10, and 20 Ag/mL) recapitulatedin a concentration-dependent manner the effects of KMKKT onPSA mRNA and protein abundance (Fig. 4B). Like KMKKT,decursin or AGN extract did not affect the AR mRNA level(Fig. 4B) but decreased AR protein abundance (Fig. 4B). Inaddition, the AGN extract and decursin induced potent G1 arrest ina concentration-dependent manner as did KMKKT (Table 1).Furthermore, treatment with decursin or AGN extract blocked theandrogen-stimulated translocation of AR to the nucleus as didKMKKT (Fig. 4C , compare lanes 5, 7 , and 9 with lane 3). Theseresults suggested that decursin in neat form or in herbal extractsinhibited AR translocation into the nucleus and decreased itsprotein abundance, preventing AR from activating PSA mRNAtranscription and protein expression.To verify this prediction of transcriptional inhibition by decursin,

we evaluated the PSA promoter-luciferase activity in a transienttransfection assay as described previously (25). As shown in Fig. 4D ,decursin treatment for 24 hours inhibited androgen-stimulatedPSA promoter transcription in a concentration-dependent manner,with IC50 of f1.6 Ag/mL (5 Amol/L).Furthermore, decursin and AGN extract recapitulated the effect

of KMKKT in the neuroendocrine differentiation assay (Fig. 5,photographed 9 days after seeding). In the absence of androgenstimulation (a-f ), decursin (e and f ), AGN extract (c and d), andKMKKT extract (b) decreased the basal PSA secretion by 4-fold( from 12 to 3 ng/mL). In the presence of androgen (g-l), decursin(k and l) and AGN extract (i and j) inhibited the androgen-stimulated reversal of neuroendocrine differentiation and androgen-stimulated cell proliferation as well as inhibited PSA secretion in

Table 1. Effects of the ethanol extracts of KMKKT, AGN, and decursin on LNCaP cell cycle distribution

Treatment Concentration (Ag/mL) Cell cycle distribution (%) Sub-G1

G1 S G2

48-h exposure

Control (DMSO) 0 71 21 8 3

KMKKT extract 20 78 14 8 450 80 12 8 3

100 87 7 6 3

24-h exposure

Control (DMSO) 0 72 20 8 1KMKKT extract 50 87 8 5 1

Mix of 10 extracts 50 85 10 5 1

A. gigas extract 5 78 14 8 1

10 81 13 6 120 91 4 4 2

Decursin 3.3 (10 Amol/L) 80 13 7 1

6.6 (20 Amol/L) 86 9 5 1

NOTE: Exponentially growing LNCaP cells were treated in the complete medium containing 10% whole serum for 24 or 48 hours before flow cytometryanalyses. Mix, a mixture of 10 individual ethanol extracts in the proportion used for the formulation of KMKKT. Sub-G1 apoptotic fraction estimation

showed no increase in cell death at concentrations tested.





a concentration-dependent manner, achieving a complete block onthese variables with 3.3 Ag/mL (10 Amol/L) of decursin and 10 Ag/mLAGN extract, respectively. The effect of a single treatment lastedthrough at least 17 days when the experiment was terminated(data not shown), indicating the long-lasting androgen suppressionaction of decursin and AGN extract. These data unequivocallysupported decursin as an antiandrogen and AR compound in AGNand KMKKT extracts.PSA suppression persists after removal of decursin or herbal

extracts. To further confirm the long-lasting action of decursin onandrogen and AR signaling, we exposed LNCaP cells for 3 days incomplete mediumwith either decursin, AGN, or KMKKTextract andthen carefully removed the conditioned medium and washed thecells twice with serum-free medium. The cells were then fed freshcomplete medium for 1, 2, or 3 days. At each time point, theconditioned medium was harvested and the cells were lysed for PSAELISAmeasurement to determine the kinetics of recovery. As shownin Fig. 6A (left), the decursin-, KMKKT-, and AGN-exposed cellsshowed minimal recovery of cellular PSA level (normalized onprotein content) at 24 hours after removal. By 3 days after removal, apartial recovery of <40% and 30% was detected for cells exposed todecursin and the two extracts, respectively. Immunoblot analysisof cellular PSA in decursin-treated cells in a separate experiment

confirmed this result (Fig. 6B). Similar patterns of persistentsuppression of PSA secreted into the medium by decursin andKMKKT or AGN extract were observed (Fig. 6A, right , and B).To determine whether the suppression of PSA expression after

the removal of decursin was due to a persistent decrease in ARabundance, we immunoblotted AR and PSA in the cell lysate(Fig. 6B). Decursin-treated cells maintained a profound suppres-sion of cellular PSA throughout 72 hours, whereas the AR proteinrebounded to control level within 24 hours of removal of decursin.These results suggested that decursin exerted a sustainedinhibitory action on androgen and AR signaling even after ARprotein abundance had fully recovered.Comparison with the effects of bicalutamide. To further

probe themodes of action of decursin on AR signaling, we comparedits effects in the presence and absence of androgen stimulation withthose of bicalutamide, which is a clinically indicated androgen-binding antagonist (26). LNCaP cells were cultured in 5% CSSmedium for 48 hours and exposed to DMSO or indicatedconcentrations of decursin or bicalutamide for 1 hour before theaddition of mibolerone for 24 hours. As shown in Fig. 6C , decursindecreased PSA mRNA and protein as well as AR protein abundancein the absence (lanes/columns 2 and 3 versus lane/column 1) andpresence (lanes/columns 7 and 8 versus lane/column 6 ) of

Figure 2. A, induction of morphologic neuriteformation in sparsely seeded LNCaP cells by KMKKT(b ) in medium containing 5% whole serum andinduction of neuroendocrine differentiation in phenolred–free medium supplemented with 5% CSS (c )and its reversal by the addition of mibolerone (d).Photomicrographs were taken 7 days after treatments.Bold arrowheads, representative neurites. The overallextent of neuroendocrine differentiation–positive(NED+) cells in each treatment was estimated byexamining at least five random fields for typical neuritefeatures. B, inhibition by KMKKT extract of theaction of androgen to suppress neuroendocrinedifferentiation. LNCaP cells were seeded into T25flasks with phenol red–free medium supplementedwith 5% CSS. KMKKT was added at seeding time at50 and 100 Ag/mL. Increasing levels of miboleronewere added 2 days after seeding. The cells werephotographed at 9 days after seeding. C, inhibitoryeffects of KMKKT extract on the androgen-stimulatedcell proliferation estimated as protein yield. D,inhibitory effects of KMKKT extract on the secretedPSA (by ELISA) accumulated in the conditionedmedium during 9 days.

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mibolerone without affecting the AR mRNA level. Bicalutamide didnot significantly change AR mRNA level either (lanes 4 and 5 versuslane 1 in the absence of mibolerone and lanes 9 and 10 versus lane 6in the presence of mibolerone). In contrast to decursin, bicalutamideincreased the basal levels of PSA mRNA and cellular and secretedPSA as well as AR abundance in the absence of androgen (lanes/columns 4 and 5 versus lane/column 1), acting as a partial AR-binding ‘‘agonist.’’ As expected of its androgen-binding blockeraction, bicalutamide decreased PSAmRNA, protein, and secretion inthe presence of mibolerone in a concentration-dependent manner(lanes/columns 9 and 10 versus lane/column 6). With respect toLNCaP survival, decursin was more potent than bicalutamide at asame molar concentration of exposure (40 Amol/L) to induceapoptosis as indicated by the cleaved PARP (lane 3 versus lane 5, lane8 versus lane 10). These results supported distinct novel mecha-nisms by which decursin inhibited androgen and AR signaling incomparison with bicalutamide.

Discussion

The work communicated here represents a beginning ofcollaborative efforts to identify novel phytochemicals that targetandrogen and AR signaling from Oriental medicinal herbs.

Identification of the active compound(s) is essential to under-standing the mechanisms of action and for developing clinicallyuseful agents for the prevention and treatment of prostate cancerand other androgen-dependent diseases, such as benign prostatichyperplasia (BPH; ref. 27) and male baldness (28). Starting fromthe ethanol extract of the KMKKT formula, we discovered potentantiandrogen and AR signaling activities, including the suppres-sion of PSA expression at both protein and mRNA levels (Fig. 1Band C) and the induction of neuroendocrine differentiation(Fig. 2) using the androgen-dependent LNCaP model. We foundthat AGN was primarily responsible for the antiandrogen and ARactivities (Fig. 3A). Solvent fractionation and TLC data (Fig. 3B)confirmed decursin as a major phytochemical of AGN extract(16, 17) and supported a direct correlation of decursin contentwith the potency of the various solvent fractions to inhibit PSAand cell growth (Fig. 3C). Treatment with purified decursinrecapitulated all the effects of KMKKT and AGN that wereexamined, including decreasing PSA protein abundance andsecretion (Fig. 4A and B) and PSA mRNA level (Fig. 4B),decreasing AR protein level without affecting its mRNAabundance (Fig. 4B), inhibiting androgen-stimulated AR transloca-tion to the nucleus (Fig. 4C) and androgen-stimulated PSA

Figure 3. A, comparison of KMKKT extract with the 10individual herbal extracts each alone (20 Ag/mL) or all 10extracts combined in the proportion making up KMKKT (Mix ) oncellular PSA expression (48-hour exposure) in LNCaP cellscultured in the complete medium. Number at the end of eachlabel, concentration (in Ag/mL) of the extract. B, TLC analysesof various solvent fractions of AGN extract. For detectionof decursin, 10% sulfuric acid was sprayed on the TLCplate to char carbon-containing compounds. Hx, n-hexane;MC, methylene chloride; EA, ethyl acetate; BuOH, butanol.C, inhibitory potency of the solvent fractions of AGN extract oncellular PSA (by ELISA) after 48-hour exposure in the completemedium. The secreted PSA was affected by the same orderof potency (data not shown). Correlation of IC50 values forsuppressing cellular PSA expression and 24-hour cell viabilityestimated by MTS assay was shown. D, chemical structure ofdecursin. The coumarin moiety is marked.





promoter transcription (Fig. 4D), suppressing androgen-stimulatedcell proliferation and overriding the ability of androgen to blockneuroendocrine differentiation (Fig. 5), and inducing a profound G1

arrest (Table 1). These results unequivocally supported decursin as apotent antiandrogen and AR compound in AGN and KMKKTextracts but did not exclude the possibility of additional activeantiandrogen and AR compounds. For example, we have foundrecently that decursinol angelate, an isomer of decursin, possesses asimilar potency for inhibiting PSA expression. Efforts are beingcontinued to identify the structural determinants of the antian-drogen and AR activities of decursin analogues and derivatives.Our data showed that the antiandrogen and AR activities were

not a consequence of apoptosis induced by decursin or by KMKKTextract. In our experiments, decursin induced apoptosis (Fig. 6C ;Supplementary Fig. S2) at exposure concentrations of z40 Amol/L(13.2 Ag/mL). This is consistent with a recent report of apoptosisinduction in LNCaP cells byz50 Amol/L (16.5 Ag/mL) decursin (20).KMKKT induced apoptosis atz200 Ag/mL concentrations (Fig. 1A).These apoptotic levels of decursin or KMKKT were much higherthan the IC50 for PSA suppression, 0.4 Ag/mL decursin (Fig. 4A) and7 Ag/mL KMKKT (Fig. 1B), respectively. The antiandrogen and ARactivities were observed in concentration ranges that caused G1

arrest (Table 1) and were likely a primary cause for the cell cyclearrest effect. This was further supported by the ability of increasingmibolerone doses to partially restore cell growth in the presence of

KMKKT (Fig. 2B and C). Because decursin has been reported toinduce G1 arrest not only in LNCaP cells but also in androgen-independent DU145 and PC-3 cells (20), decursin could thereforeexert antiproliferative effects through both androgen/AR–dependentand androgen/AR–independent mechanisms, the details of whichremain to be elucidated. The antiandrogen and AR activities couldbe predicted to make decursin more active against androgen-dependent prostate cancer cells than androgen-independent cellsfor growth inhibition and apoptosis. Consistent with this, we found(Supplementary Fig. S2) that PC-3 cells were less sensitive thanLNCaP cells for decursin-induced apoptosis, in agreement with arecent report (20).Our results reveal at least two possible mechanisms through

which decursin may exert the antiandrogen and AR activities:inhibiting androgen-stimulated AR nuclear translocation anddecreasing AR protein abundance. With respect to the firstmechanism, it remains to be determined whether decursincompetes with androgen for binding to AR to prevent it fromtranslocating to the nucleus to activate transcription of AR-responsive genes, such as PSA (Fig. 4D). A comparison withbicalutamide has revealed significant differences between theiractions, particularly under androgen-free conditions (Fig. 6C). Thatis, bicalutamide increased (instead of inhibited) basal AR proteinand PSA abundance and secretion in contrast to decursin,which suppressed these variables. In addition, decursin was

Figure 4. A, concentration-dependent inhibition bypurified decursin of cellular and secreted PSA(detected by ELISA) in LNCaP cells after 48-hourexposure in the complete medium. B, RT-PCRdetection (mRNA) and immunoblot detection (protein)of effects of decursin (lanes 6-8 ), AGN extract(lanes 3-5), and KMKKT (lane 2) on PSA and ARlevels after exposure for 24 hours in the completemedium. C, immunoblot detection of effects ofdecursin, AGN, and KMKKT extracts on AR nucleartranslocation in LNCaP cells. LNCaP cells were grownin phenol red–free medium supplemented with 5%CSS for 2 days. The cells were pretreated 100 Ag/mLKMKKT extract (K100 ), 10 Ag/mL AGN (AGN10 ), or3.3 Ag/mL decursin (D3.3) for 24 hours and werestimulated with 0.1 nmol/L mibolerone for 6 hours inthe continued presence of KMKKT extract (totalexposure time of 30 hours). Nuclear and cytosolicfractions were prepared for immunoblot analyses.PARP and tubulin were detected as markers of nuclearand cytosol proteins, respectively. D, effect ofdecursin on androgen-stimulated PSA promoteractivity. LNCaP cells were cotransfected with PSApromoter-luciferase and CMV-h-gal vectors andincubated in serum-free, phenol red–free medium for24 hours. The cells were treated with decursin + 1nmol/L mibolerone for 24 hours in 5% CCS-addedmedium. Cell lysates were assayed for luciferaseand h-gal activities. Luciferase activities werenormalized to h-gal activities for correcting transfectiondifferences. Results were based on twoexperiments, each with duplicate wells per treatmentconcentration.

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more potent than a same molar concentration of bicalutamide forinduction of apoptosis (Fig. 6C). These results support decursin asa novel and potent antiandrogen and AR agent with severalaspects of mechanisms of action distinct from bicalutamide. Withrespect to the second mechanism, because AR protein is down-regulated by decursin, AGN, and KMKKT extracts, but the ARmRNA level was not affected (Figs. 1B , 4B , and 6C), we arecurrently investigating whether AR protein synthesis and/ordegradation are targeted by decursin. Furthermore, decursin hasbeen reported as an activator of protein kinase C (PKC) in testtube assays and to inhibit leukemia differentiation induced bysome tumor-promoting PKC activators (17, 18). Because PKCactivators, such as 12-O-tetradecanoylphorbol-13-acetate andphorbol-12-myristate-13-acetate, have been reported to suppressPSA (29, 30) and induce apoptosis in LNCaP cells (30, 31), whetherthe antiandrogen and AR activities of decursin in LNCaP cells aremediated by PKC pathway should be examined in the future.One salient feature of the antiandrogen and AR activities of

decursin or AGN and KMKKTextracts is the long-lasting action. ThePSA-suppressing effect of a single treatment by KMKKTwas initiallymanifested at 9 hours of exposure with the lowering of PSA mRNA(Fig. 1C) and then of cellular PSA protein at 12 hours of exposure(Fig. 1B) and persisted for 96 hours without any indication ofrecovery (Fig. 1B and C). Similarly and more strikingly, we observedthat neuroendocrine differentiation persisted for 2 to 4 weeks with asingle treatment with decursin or the herbal extracts (Figs. 2 and 5).The long-lasting action was further evident when the treatments

were removed with only minor partial recovery of cellular PSAprotein 3 days afterward (Fig. 6A and B). The lasting action afterdecursin removal did not result from a continually suppressed ARprotein abundance because it rebounded to control level 24 hoursafter decursin was removed (Fig. 6B). We are currently investigatingwhether this persistent suppression of PSA might be due to a sus-tained blockage of AR nuclear translocation or an epigenetic inacti-vation of the transcribability of the PSA promoter. If translatablepharmacologically to in vivo , the long-lasting action of decursinwould have a significant advantage in terms of the dosing frequencyto maintain an effective suppression of androgen and AR activity.The identification of decursin as a novel antiandrogen and AR

signaling compound at low concentration levels (<10 Amol/L)provides specific mechanistic rationales for its potential use in thechemoprevention of androgen-dependent prostate cancer and forthe treatment of BPH (27) and male baldness (28). This discoveryand the recent report (20) of induction by decursin of G1 arrest inDU145 and PC-3 AR-negative and androgen-independent prostatecancer cells at 25 Amol/L (8.2 Ag/mL) and apoptosis at 100 Amol/L(32.8 Ag/mL) suggest that decursin could also be useful for thechemoprevention or treatment of hormone-refractory prostatecancer. Regarding the latter, recent studies have shown that even inhormone-refractory prostate cancer AR is mostly present in wild-type status and that ligand-independent AR signaling is importantfor the growth and survival of these prostate cancer cells (2, 32, 33).The ability for decursin to suppress AR abundance and signaling inthe absence of androgen (Fig. 6C) could therefore be important for

Figure 5. Comparison of the effects of decursin and AGN extract with KMKKT extract on the androgen-stimulated cell proliferation and suppression of neuroendocrinedifferentiation at 9 days after seeding LNCaP cells with designated treatments in phenol red–free medium containing 5% CCS. Mibolerone was added to flasks(g-l ) at 2 days after seeding the cells. Secreted PSA was measured by ELISA.





the management and prevention of hormone-refractory prostatecancer in combination with hormone ablation therapies. Animalmodels of prostate cancer chemoprevention and therapy are clearlywarranted to establish the efficacy of decursin and herbal extractsbefore any translational research into human populations iscontemplated.

AcknowledgmentsReceived 5/31/2005; revised 9/23/2005; accepted 10/12/2005.

Grant support: The Hormel Foundation, Eagles’ Telethon, National CancerInstitute grant CA95642 ( J. Lu), and Korean Biogreen21 Project grant1000520030030000-1, Korean Ministry of Health and Welfare grant 01-PJ9-PG1-01CO05-0004, and Interdisciplinary Research and SRC Program of Korea Science andEngineering Foundation (S-H. Kim).

The costs of publication of this article were defrayed in part by the payment of pagecharges. This article must therefore be hereby marked advertisement in accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.

We thank Dr. Nam-In Baek for authenticating the herbs used in this study, Prof.Hogyu Han for the crystalline decursin sample, Dr. Charles Young for the generousgifts of PSA promoter, CMV-lacZ plasmids, and mibolerone, Dr. Hongbo Hu for helpwith apoptosis ELISA assay, Todd Schuster for flow cytometry analyses, and AndriaHanson for secretarial support.

Figure 6. A, persistence ofPSA-suppressing effect ofKMKKT (100 Ag/mL), AGN(20 Ag/mL), and decursin (6.6 Ag/mL or 20 Amol/L) after removal.Left, cellular PSA (by ELISA) wasnormalized to protein yield; right,secreted PSA. LNCaP cells weretreated for 3 days in completemedium with one of the threetreatments. After removal ofconditioned medium, the cellswere carefully washed. The cellswere fed fresh complete mediumfor 1, 2, and 3 days. Theconditioned medium washarvested and the cells werelysed for PSA measurement todetermine the kinetics ofrecovery. B, immunoblotdetection of cellular PSA and ARabundance at 1, 2, and 3 daysafter removal of decursin(30 Amol/L). The correspondingsecreted PSA values werepresented below each lane. Thetreatment protocol was same asin (A ). C, comparison of potencyand manners by which decursinand bicalutamide affected PSAand AR mRNA detected byRT-PCR as well as cellular andsecreted PSA, AR proteinabundance, and PARP cleavageas a marker of apoptosis inthe absence and presence ofmibolerone stimulation for24 hours.

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2006;66:453-463. Cancer Res Cheng Jiang, Hyo-Jeong Lee, Guang-xun Li, et al. Cancerwith Implications for Prevention and Treatment of ProstateIdentification of Decursin as a Novel and Active Compound

Containing Herbal Formulation:−Angelica gigasPotent Antiandrogen and Androgen Receptor Activities of an

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