Journal of Cellular Biochemistry 104:1612–1624 (2008)

5a-Androstane-3a,17b-Diol Supports Human ProstateCancer Cell Survival and Proliferation Through AndrogenReceptor-Independent Signaling Pathways: Implicationof Androgen-Independent Prostate Cancer Progression

Qing Yang,1 Mark A. Titus,2 Kar-Ming Fung,3,4 and Hsueh-Kung Lin1,4*1Department of Urology, University of Oklahoma Health Sciences Center, Oklahoma City,Oklahoma 731042Urologic Oncology, Roswell Park Cancer Institute, Buffalo, New York 142633Department of Pathology, University of Oklahoma Health Sciences Center, Oklahoma City,Oklahoma 731044Department of Veterans Affairs Medical Center, Oklahoma City, Oklahoma 73104

Abstract Androgen and androgen receptor (AR) are involved in growth of normal prostate and developmentof prostatic diseases including prostate cancer. Androgen deprivation therapy is used for treating advanced prostatecancer. This therapeutic approach focuses on suppressing the accumulation of potent androgens, testosterone and5a-dihydrotestosterone (5a-DHT), or inactivating the AR. Unfortunately, the majority of patients with prostate cancereventually advance to androgen-independent states and no longer respond to the therapy. In addition to the potentandrogens, 5a-androstane-3a,17b-diol (3a-diol), reduced from 5a-DHT through 3a-hydroxysteroid dehydrogenases(3a-HSDs), activated signaling may represent a novel pathway responsible for the progression to androgen-independentprostate cancer. Androgen sensitive human prostate cancer LNCaP cells were used to compare 5a-DHT and 3a-diolactivated androgenic effects. In contrast to 5a-DHT, 3a-diol regulated unique patterns of b-catenin and Akt expression aswell as Akt phosphorylation in parental and in AR-silenced LNCaP cells. More significantly, 3a-diol, but not 5a-DHT,supported AR-silenced LNCaP cells and AR negative prostate cancer PC-3 cell proliferation. 3a-diol-activated androgeniceffects in prostate cells cannot be attributed to the accumulation of 5a-DHT, since 5a-DHT formation was not detectedfollowing 3a-diol administration. Potential accumulation of 3a-diol, as a result of elevated 3a-HSD expression incancerous prostate, may continue to support prostate cancer growth in the presence of androgen deprivation. Futuretherapeutic strategies for treating advanced prostate cancer might need to target reductive 3a-HSD to block intraprostatic3a-diol accumulation. J. Cell. Biochem. 104: 1612–1624, 2008. � 2008 Wiley-Liss, Inc.

Key words: androgen; androgen receptor; cell growth; prostate cancer

The human prostate is an androgen sensitiveorgan that depends on androgen for growth anddevelopment [Coffey and Isaacs, 1981]. Potentandrogens such as testosterone and 5a-dihy-drotestosterone (5a-DHT) as well as theircorresponding nuclear receptor (androgenreceptor; AR) have been heavily implicated inthe development of normal prostate and pro-static diseases including prostate cancer [Voigtand Bartsch, 1986; Berger et al., 2004]. Currenttherapeutic strategies for treating patients withadvanced prostate cancer, therefore, mainlyrely on androgen deprivation [Sharifi et al.,2005] to suppress the intraprostatic accumula-tion of potent androgens or to inactivate AR

� 2008 Wiley-Liss, Inc.

Abbreviations used: 3a-diol, 5a-androstane-3a, 17b-diol;3a-HSD, 3a-hydroxysteroid dehydrogenase; 5a-DHT, 5a-dihydrotestosterone; AKR, aldo-keto reductase; AR, andro-gen receptor; ARG, androgen responsive gene; siRNA,small interfering RNA; TUNEL, terminal deoxynucleotidyltransferase biotin-dUTP nick end labeling.

Grant sponsor: US Department of Veterans Affairs MeritAward.

*Correspondence to: Hsueh-Kung Lin, PhD, Department ofUrology, University of Oklahoma Health Sciences Center,920 Stanton L. Young Blvd., Oklahoma City, OK 73104.E-mail: hk-lin@ouhsc.edu

Received 9 July 2007; Accepted 18 January 2008

DOI 10.1002/jcb.21731

trans-activation [Seidenfeld et al., 2000; Smithet al., 2004; Tay et al., 2004]. However, the vastmajority of patients who initially respond to thetherapy inevitably progress to highly aggres-sive, androgen-independent prostate cancer[Daneshgari and Crawford, 1993]. The mecha-nisms by which prostate cancer cells survive inandrogen deprivation and progress to an andro-gen-independent state remain unclear.

The potent androgen 5a-DHT can be reducedto 5a-androstane-3a, 17b-diol (3a-diol) by agroup of isozymes named 3a-hydroxysteroiddehydrogenases (3a-HSDs) in the prostate. Two3a-HSD isozymes, type 2 3a-HSD (AKR1C3)and type 3 3a-HSD (AKR1C2), have beenidentified in the human prostate [Penninget al., 2000]. Both 3a-HSDs possess dominant5a-DHT reduction activity toward 3a-diol for-mation [Lin et al., 1997; Rizner et al., 2003;Penning et al., 2007]. In addition, elevatedlevels of 3a-HSD expression have been demon-strated in primary cultures of prostate epithe-lial cells derived from prostate cancer [Lin et al.,1997; Rizner et al., 2003] and in prostate cancertissues [Nakamura et al., 2005; Fung et al.,2006; Stanbrough et al., 2006]. These resultsstrongly suggest that 3a-diol can be accumu-lated in cancerous prostate.

Based on 3a-diol’s low affinity toward the AR,3a-diol has been recognized as a weak androgenand does not have androgenic effects [Baumanet al., 2006]. However, this assumption does notexplain why 3a-diol is more potent than 5a-DHTor testosterone when inducing prostatic hyper-plasia in castrated dogs [Walsh and Wilson,1976; Jacobi et al., 1978]. 3a-diol is also an activeandrogen in virilizing the urogenital tract offemale rat embryos [Schultz and Wilson, 1974],plays a significant role in murine parturition[Mahendroo et al., 1996], and is implicated inprostate and penile development of femalefetuses of marsupials [Shaw et al., 2000; Leihyet al., 2004]. In addition, Agapova et al. [2006]demonstrated that 3a-diol is responsible foroptic nerve head astrocyte mobility and sur-vival. Growing evidence suggests that 3a-diolmight be an important androgen with its ownfunctions in androgen target tissues.

The mechanism of 3a-diol-activated andro-genic effects remains undefined. Ding et al.[1998] demonstrated that 3a-diol forms acomplex with sex hormone-binding globulin(SHBG) at the cell surface of androgen targettissues to stimulate rapid accumulation of

intracellular cAMP with subsequent activationof the AR. However, 3a-diol-activated signalingmay be different from the classical AR-mediatedsignaling pathway in prostate cells. Nunlistet al. [2004] reported that 3a-diol is as potentas 5a-DHT in stimulating androgen sensitivehuman prostate cancer LNcaP cell prolifera-tion, but does not execute similar levels of ARtrans-activation. In addition, 3a-diol-regulatedgenes can be distinguished from 5a-DHT-regulated androgen responsive genes (ARGs)[Zimmerman et al., 2004]. The present studyemphasizes that 3a-diol can activate AR-independent ARG expression and cytoplasmicsignaling, as well as promote prostate cell sur-vival and proliferation. These results suggestthat potential intraprostatic accumulation of3a-diol through elevated 3a-HSD expression incancerous prostate might activate cell survivalpathway and support androgen-independentcancer progression in the presence of androgendeprivation therapy.

MATERIALS AND METHODS

Materials

RPMI 1640 medium, F-12 nutrient mixture,OPTI-MEM, penicillin-streptomycin, and fetalbovine serum (FBS) were purchased from In-vitrogen (Grand Island, NY). Charcoal-dextrantreated (CD) FBS with testosterone <10�10 Mwas obtained from HyClone (Logan, UT). Pre-synthesized AR-specific and control SMART-pools1 small interfering RNA (siRNA) duplexeswere obtained from Upstate (Charlottesville,VA). Mouse anti-human AR monoclonal anti-body was obtained from Novocastra (UK).Rabbit anti-Akt (or protein kinase B; PKB)antibody, rabbit anti-phospho-Akt Ser(473)antibody, and rabbit anti-b-catenin antibodywere purchased from Cell Signaling Technology(Danvers, MA). Mouse anti-b-actin antibodywas obtained from Sigma (St. Louis, MO). 3a-diol and 5a-DHT were obtained from Steraloids(Newport, RI).

Plasmid Constructs

Plasmid-based AR-specific and nonspecificcontrol siRNA constructs were modified fromour previous report with the excision of theIRES sequence [Yang et al., 2005b]. Theseconstructs were designated as pSiAR-EGFPand pSiCon-EGFP for AR-specific and controlsiRNA plasmids, respectively. These siRNA

3a-Diol-Activated AR Independent Pathway 1613

plasmids contain enhanced green fluorescenceprotein (EGFP) driven by the cytomegalovirus(CMV) promoter. Transfected cells constitu-tively expressing EGFP allowed for rapidenrichment.

Human Prostate Cell Cultureand Transfection

Androgen sensitive human metastatic pros-tate adenocarcinoma LNCaP cells (CRL-1740)and human bone metastasized prostate cancerPC-3 cells (CRL-1435) were obtained fromATCC (Manassas, VA). LNCaP cells weremaintained in a complete growth medium(RPMI1640 supplemented with 10% FBS)[Nunlist et al., 2004] and used at low passagenumbers (less than 35 passages in total). PC-3cells were maintained in F-12 nutrient mixturecontaining 7% FBS.

Suppression of the endogenous AR expressionin LNCaP cells was achieved using two siRNAapproaches: an oligo-based siRNA duplex anda plasmid-based siRNA construct. The oligo-based SMARTpools1 siRNA duplex was used toanalyze androgen-stimulated ARG expressionand cytoplasmic signal activation based on itshigh transfection efficiency. Briefly, trypsinizedLNCaP cells (2� 106) were mixed with 20 mMeither SMARTpools1 AR-specific or controlsiRNA duplex in 100 ml of Nucleofector solutionR (Amaxa Biosystems, Gaithersburg, MD); andtransfection was completed with the Nucleo-fector device (Amaxa Biosystems). Cells werethen returned to their growth medium in 60 mmtissue culture plates for 24 h before serumdeprivation (OPTI-MEM plus 2% CD FBS) andandrogen stimulation.

The siRNA plasmid was used to determineandrogen-stimulated cell proliferation and sur-vival based on its sustained suppression of ARexpression in prostate cells. Briefly, LNCaPcells were transfected with either pSiAR-EGFPor pSiCon-EGFP plasmid with Lipofectamine2000 (Invitrogen) using our reported proce-dures [Yang et al., 2005b]. Cells were thenreturned to their growth medium with or with-out androgen supplementation at 4 h followingtransfection.

Androgen Stimulation

To determine androgen conversion, LNCaPcells (5� 105) were seeded in the growthmedium in six-well tissue culture plates for

adherence. Cells were subjected to serumdeprivation for 24 h and switched to 1 ml serumfree OPTI-MEM medium for another 4 h. Cellswere left untreated or treated with either 10�8 M5a-DHT (positive control) or 3a-diol. Sampleswere collected at 24 h following androgen treat-ment by harvesting the cells and mediatogether, immediately frozen with dry ice inacetone, and stored at �808C until analysis.

To study androgens-stimulated ARG expres-sion and Akt phorphorylation, parental andsiRNA duplex transfected LNCaP cells (1� 106)were first seeded in 60 mm tissue culture platesfor adherence. Cells were then subjected toserum deprivation for 24 h followed by 10�8 Mandrogen stimulation.

Androgen-stimulated prostate cell prolife-ration was performed in both LNCaP and PC-3cells. At 24 h following pSiAR-EGFP or pSiCon-EGFP transfection, EGFP expressing LNCaPcells (1,000) were directly distributed into eachwell of a 96-well tissue culture plate containing200 ml of their growth medium with or withoutandrogens using MoStar cell sorter [Yang et al.,2005b]. PC-3 cells (1,000 cells in 100 ml) werealso seeded in each well of a 96-well plate intheir growth medium, and were either leftuntreated or treated with 3a-diol. For cell deathanalysis, siRNA plasmid transfected LNCaPcells were either left untreated or treated withandrogens.

5a-DHT Measurement

LC/MS/MS detection of 5a-DHT was per-formed as described [Titus et al., 2005] withmodifications. Deuterated testosterone and 5a-DHT (1 ng each) were added to thawed LNCaPcells with media as internal standards; andsamples were extracted three times with 1 mlof 9:1 chloroform/acetone. Collected organicswere evaporated under vacuum and analytesconcentrated using solid phase extraction car-tridges (SPEC-C18AR, Varian) conditioned withmethanol and water. Samples were applied in1:4 methanol/water. Samples were eluted inmethanol, dried under vacuum, and reconsti-tuted in 65% methanol for analysis.

The LC/MS/MS system used was the ThermoFinnnagan Surveyor quaternary MS pump,solvent degasser, and a Surveyor auto samplerinterfaced with TSQ Quantum ULTRA massspectrometer. A Phenomenex Luna C18 (3 mm,150� 2.0 mm) column was used to separatetestosterone and 5a-DHT. The column was

1614 Yang et al.

maintained at 308C. An HPLC gradient profilewas used from 65% to 75% methanol in 2.25 minfollowed: 60% B from 0.0 to 1.0 min, 70% to 100%B from 1.1 to 9.0 min, 100% B from 9.0 to14.0 min, 100–60% B from 14.0 to 14.5 min. Thecolumn was equilibrated at 60% B for 12 minprior to sample injection.

Testosterone and 5a-DHT were ionized usingan atmospheric pressure chemical ionizationsource in positive ion mode and were quantifiedusing unique product ions. The parent/production pairs of m/z 289.2 to 97.0 for testosterone,m/z 292.2 to 97.0 for internal standard testo-sterone-d3, m/z 291.2 to 255.1 for 5a-DHT, andm/z 294.2 to 258.1 for internal standard 5a-DHT-d3 were monitored and the product ionwas used to quantify androgens. Optimizedcollision energy settings for testosterone, tes-tosterone-d3 and 5a-DHT, 5a-DHT-d3 were 25,18 V, respectively. Other mass spectrometryparameters were collision gas pressure 1.2 mTorr, discharge current 4.0 kV, vaporizingtemperature 4808C, sheath gas 30 Arb, Auxil-iary gas 7 Arb, capillary temperature 2808C.Nitrogen was used for all gas inputs.

Western Blot Analysis

To determine levels of target protein expres-sion, total cellular proteins were isolated bylysing cells with RIPA buffer that was supple-mented with 0.1 mM phenylmethylsulphonyl-fluoride (PMSF) and a proteinase inhibitorcocktail (Roche, Indianapolis, IN) [Yang et al.,2005b]. Protein concentrations were deter-mined by a bicinchoninic acid (BCA) proteinassay kit (Pierce, Rockford, IL). Aliquots of 30mgof the cellular proteins were separated on 10%Tris–HCl gel (Bio-Rad, Hercules, CA); andproteins were transferred to PVDF membranes(Bio-Rad). Detection of the AR and b-actinexpression was performed as reported [Yanget al., 2005b]. b-catenin, total Akt, and phospho-Akt expression were detected by incubatingthe PVDF membranes with primary antibodiesagainst these molecules followed by appropriateperoxidase-conjugated secondary antibodyincubation. Immunoreactive proteins weredetected using an enhanced chemiluminescent(ECL) reagent (Pierce). To quantify levels of thetarget protein expression, images of immunor-eactive bands were captured; and intensities ofthese bands were calculated using the compu-terized Quantity One1 image analysis software(Bio-Rad).

Cell Proliferation Assay

Cell proliferation was performed directly in96-well tissue culture plates using a XTT cellproliferation assay kit (Roche). Briefly, analiquot of 50 ml XTT labeling mixture was addedto each well of the 96-well plates containing100 ml of culture medium and incubated at 378Cfor 4 h. Absorbance was obtained by scanningthe plates using mQuant microplate reader (Bio-Tek, Winooski, VT). Data normalization andanalysis was performed as previously reported[Nunlist et al., 2004].

Terminal Deoxynucleotidyl TransferaseBiotin-dUTP Nick End Labeling (TUNEL) Analysis

TUNEL analysis was performed using IHC-like staining procedures in cell block sections.Briefly, on day 6 after siRNA plasmid trans-fection and androgen stimulation, LNCaP cellswere trypsinized and collected through centri-fugation. Cell pellets were fixed in 10% formalin,immersed in agarose, and subjected to paraffinembedding. The cell blocks were sectioned,dewaxed, and rehydrated [Yang et al., 2005b].Cells were permeabilized by incubating thesections with 20 mg/ml proteinase K at roomtemperature for 20 min; and DNA fragmenta-tion was detected using an in situ Cell DeathDetection kit (Roche). Positive control was pre-pared by digesting parental LNCaP cell sectionswith 1 U/ml DNase I at room temperature for10 min. Following the terminal deoxynucleo-tidyl transferase reaction, cell sections wereincubated with the converter-alkaline phospho-tase. A Fast Red substrate was added to theslides and incubated at room temperature for anadditional 10 min for color development. Slideswere then washed and sealed with an aqueousmounting medium. Apoptotic cells with dam-aged DNA were stained positive with a brightred color.

To quantify the number of apoptotic cells,five sections from each experimental group werestudied. On each slide, three fields were ran-domly picked; and images were taken at 200�magnification by Olympus BX51 microscopeequipped with SPOT Insight CCD digitalcamera and SPOT Advance software (Diagnos-tic Instruments; Sterling Heights, MI). Totalnumber of cells and red cells were counted foreach image. The mean and standard deviationof the percentage of TUNEL positive cells foreach experimental group were calculated.

3a-Diol-Activated AR Independent Pathway 1615

Statistical Analysis

Statistical differences between two experi-mental groups were evaluated using studentt-test. Statistically significant difference wasset when P< 0.01.

RESULTS

3a-Diol and 5a-DHT Induced Differential ARGExpression and Cytoplasmic Signaling

Nunlist et al. [2004] reported that b-cateninand Akt respond to both 3a-diol and 5a-DHTstimulation in LNCaP cells; but temporalregulation of steady state levels of b-cateninand Akt mRNA expression was differentbetween 3a-diol and 5a-DHT treatments. West-ern blot analysis confirmed that in spite ofinitial (within 30 min) suppression of b-cateninexpression by both androgens, 5a-DHT-elevated

b-catenin expression was almost immediateand sustained for 24 h. In contrast, recovery ofinitial 3a-diol-suppressed b-catenin expressionwas slower and extended to 8 h (Fig. 1A,B); andlevels of b-catenin in 3a-diol-treated LNCaPcells were doubled as compared to untreatedcontrol at 24 h. In addition, 5a-DHT-stimulatedAkt protein expression peaked at 30 min butgradually subsided (Fig. 2A,B). On the contrary,3a-diol suppressed Akt expression between30 min and 4 h; but elevated Akt expressionwas detected at 8 h post 3a-diol treatment(Fig. 2A,B).

5a-DHT regulated Akt phosphorylaiton ina time-dependent manner. However, 3a-diolhad more potent and sustained effects on Aktphosphorylation in LNCaP cells as compared tothe same concentration of 5a-DHT (Fig. 2A).Semi-quantitative analysis of Akt phosphoryla-tion levels demonstrated that 3a-diol induces

Fig. 1. Temporal regulation ofb-catenin expressionby 3a-diol and 5a-DHT in LNCaPcells. A: Western blotanalysis of androgen-regulated b-catenin expression between 30 min and 24 h in LNCaP cells. B: Semi-quantitative levels of b-catenin expression between 3a-diol and 5a-DHT treated LNCaP cells. Intensities ofimmunoreactive bands were first normalized to levels of b-actin within each time point; and levels of thenormalized values were then adjusted to untreated cells for each androgen treatment. Experiments wererepeated at least twice and a representative result was presented.

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Akt phosphorylation by about twice as much as5a-DHT treatment at all time points (Fig. 2C).

AR-Specific siRNA DuplexSuppressed AR Expression

To investigate the involvement of the ARin androgen actions, RNA interference (RNAi)technology was introduced to suppress endo-genous AR expression in LNCaP cells. Todetermine the efficiency of an AR-specificsiRNA duplex in suppressing AR expression,LNCaP cells were transfected with an AR-specific or control SMARTpools1 siRNA duplex.Western blot analysis demonstrated that theAR-specific siRNA duplex successfully silencesAR expression in LNCaP cells between 24 and72 h following transfection (Fig. 3). Althougha higher transfection efficiency was achievedusing the duplex siRNA as compared to plas-mid-based siRNA construct, gene silencing bythe duplex siRNA was transient and the ARre-expressed in LNCaP cells after 72 h post-

transfection (data not shown). As demonstratedby Yang et al. [2005b] AR protein expressioncan be suppressed in LNCaP cells transfectedwith an AR-specific siRNA plasmid; and thesuppressed AR expression can be sustained forat least 6 days.

5a-DHT Accumulation in 3a-Diol-TreatedProstate Cells

Limits of detection for testosterone and5a-DHT using LC/MS/MS were 8.7 and 43 fmol,respectively. 5a-DHT was only detected inpositive control samples at 0.4 nM but not in3a-diol-treated LNCaP cells or negative controlsamples. However, testosterone was detected inall samples but was below the limit of quanti-fication.

3a-Diol Activated AR-Independent ARGExpression and Akt Signaling

Transfection of nonspecific siRNA duplex didnot alter androgen-regulated ARG expression

Fig. 2. Temporal regulation of Akt expression and phosphorylation in androgen-stimulated LNCaP cells.A: Western blot analysis was performed using total cellular proteins obtained from either 3a-diol or 5a-DHTtreated LNCaP cells. B: Semi-quantitative presentation of Akt expression was first normalized to b-actinwithin each time point and then adjusted to normalized values of untreated cells. C: Semi-quantitative levelsof Akt phosphorylation. Experiments were repeated at least twice and a representative result was presented.

3a-Diol-Activated AR Independent Pathway 1617

or Akt phosphorylation as compared to untrans-fected parental LNCaP cells (data not shown).3a-diol continued to regulate both b-catenin andAkt protein expression in a temporal mannerin AR-silenced LNCaP cells (Fig. 4). 3a-diol-regulated b-catenin expression exhibited sim-ilar temporal changes with and without theAR; levels of b-catenin accumulation were sup-pressed at 30 min but elevated at 24 h following3a-diol stimulation. In the absence of theAR, levels of total Akt expression were highlyelevated between 1 and 24 h and peaked at 4 hfollowing 3a-diol treatment (Fig. 4A,B). 3a-diolwas also capable of stimulating Akt phosphory-lation in LNCaP cells in an AR-independentmanner; a twofold induction in Akt phosphory-lation levels was observed at 24 h after3a-diol treatment in AR-silenced LNCaP cells(Fig. 4A,B).

3a-Diol Promoted Prostate CellProliferation and Survival

Similar to reports by others, LNCaP cellsrequired a functional AR for their proliferationin their growth medium (Fig. 5A). Supplemen-tation of 5a-DHT did not support AR-silencedLNCaP cell proliferation (Fig. 5A). In contrast,the addition of 3a-diol partially restored LNCaPcell proliferation in the absence of the AR. Cellnumber increased by 4.2- and 6.4-fold at 11 and14 days in 3a-diol-treated AR-silenced LNCaPcells as compared to 1.2- and 1.7-fold increasesin untreated cells during the same time periods.However, 3a-diol-stimulated AR-silenced LNCaPcell proliferation was not as rapid as pSiCon-EGFP-transfected LNCaP cells during theentire course of the experiments (Fig. 5A). Inaddition, 3a-diol significantly promoted ARnegative PC-3 cell proliferation as compared to

untreated cells in their growth medium(Fig. 5B).

The lack of cell proliferation in the AR-silenced LNCaP cells might result from elevatedcell death. AR silencing in LNCaP cells resultedin apoptosis as demonstrated by an increasednumber of TUNEL positive cells compared tocontrol siRNA plasmid-transfected LNCaP cells(Fig. 6 and Table I). 5a-DHT supplementationin the complete growth medium did not reducethe number of TUNEL positive cells (data notshown). In contrast, 3a-diol-treated AR-silencedLNCaP cells had significantly reduced number

Fig. 3. Suppression of AR expression using the SMARTpools1

siRNA duplex. Western blot analysis was used to determinethe successful suppression of the AR in LNCaP cells. There wasno detectible AR protein expression in LNCaP cells between24 and 72 h following the AR-specific siRNA duplex transfection.In contrast, levels of AR expression were not affected in cellstransfected with the control siRNA duplex.

Fig. 4. Temporal regulation of 3a-diol-stimulated ARGexpression and Akt phosphorylation in AR-silenced LNCaP cells.A: Levels of b-catenin and Akt expression as well as Aktphosphorylation were determined in LNCaP cells following theAR-specific siRNA duplex transfection and 3a-diol stimulationusing Western blot analysis. B: Semi-quantitative presentation of3a-diol-regulated ARG expression and Akt phosphorylation wasperformed by first normalizing to levels of b-actin expression andthen to untreated controls. Experiments were repeated at twiceand a representative result was presented.

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of TUNEL positive cells as compared tountreated AR-silenced LNCaP cells (Table I).

DISCUSSION

This study investigated whether or not 3a-diol-activated signaling can be distinguishedfrom the classical AR-mediated pathway, andwhether or not 3a-diol can activate cell survivalsignaling in prostate cells. Our results demon-strated that 3a-diol can have unique androgeniceffects on ARG expression and cytoplasmic

signaling independent from the AR pathway.More importantly, in contrast to 5a-DHT,3a-diol can support human prostate cancer cellsurvival and proliferation in the absence ofthe AR.

3a-diol has been generally classified as aweak androgen based on its low affinity towardthe AR. Therefore, 3a-diol-mediated androgeniceffects have been attributed to its oxidizedproduct, 5a-DHT, through oxidative 3a-HSDs[Bauman et al., 2006]. This pathway is sup-ported by the evidence that 3a-diol’s androgeniceffects in prostate bud formation of femaleTammar Wallaby pouch young can be blockedby the administration of antiandrogens [Leihyet al., 2001]. However, if 3a-diol needs to beoxidized to 5a-DHT to exert its androgeniceffects, it is difficult to explain why 3a-diolstimulates robust LNCaP cell proliferation[Nunlist et al., 2004] without detectible 5a-DHT accumulation [Rizner et al., 2003].

Mechanisms that are responsible for 3a-diolto exert its androgenic effects remain unde-fined. Based on gene expression profiling anal-ysis, 3a-diol responsive genes are differentfrom 5a-DHT responsive genes in LNCaP cells[Nunlist et al., 2004; Zimmerman et al., 2004].b-catenin and Akt were two of the ARGs whosesteady state levels of mRNA are differentiallyregulated by 3a-diol and 5a-DHT. b-cateninis a multifunctional protein involved in twoapparently independent processes, cell–celladhesion and signal transduction, throughWnt signaling pathway [Aguilera et al., 2007].The canonical Wnt signaling pathway modu-lates androgen signaling at multiple levels; andb-catenin represents a major molecule associ-ated with the AR [Sharma et al., 2002]. Thephosphoinositide 3 kinase (PI3K)/Akt signalingis recognized as a major cell proliferation andsurvival pathway in a variety of cells [Chinniand Sarkar, 2002; Chang et al., 2003]. Bothb-catenin and Akt signaling pathways havebeen implicated in the development of andro-gen-independent prostate cancer [Terry et al.,2006; Xin et al., 2006; Wu and Huang, 2007].

To compare 3a-diol- and 5a-DHT-activatedandrogenic effects in LNCaP cells, our resultsconfirmed that b-catenin and Akt proteinexpression as well as Akt phosphorylation aredifferentially regulated by both androgens.Consistent with published results [Sun et al.,2003; Kang et al., 2004; Gatson et al., 2006],5a-DHT activates nongenomic signaling such as

Fig. 5. 3a-diol-supported prostate cell proliferation. A: LNCaPcell proliferation in the absence of the AR. LNCaP cells weretransfected with the AR-specific or nonspecific siRNA plasmid.The transfected cells were left untreated or treated with either 3a-diol or 5a-DHT in their growth medium and sorted into 96-welltissue culture plates for cell proliferation assay. Number of viablecells was determined at designated time points using the XTT cellproliferation assay kit. B: 3a-diol-stimulated PC-3 cell prolife-ration. Human prostate cancer PC-3 cells were seeded in 96-welltissue culture plates in their growth medium followed by 3a-dioltreatment. Cell number was determined by the XTT assay for aperiod of 6 days. Asterisk (*) indicates statistical significancebetween untreated and 3a-diol-treated cells (P<0.01). Allexperiments were prepared in triplicate; and data were presentedas mean� standard error of means (SEM) from at least threeindependent experiments.

3a-Diol-Activated AR Independent Pathway 1619

Akt phosphorylation in LNCaP cells. The lowerlevels of Akt phosphorylation following 5a-DHTstimulation compared to other reports mayreflect the passage-dependent PI3K/Akt activa-

tion in LNCaP cells [Lin et al., 2003]. If 3a-diolneeds to be oxidized to 5a-DHT before it canexert its androgenic actions in prostate cells,3a-diol-regulated ARG expression and Akt

Fig. 6. Evidence of 3a-diol-induced LNCaP cell survival in the absence of the AR. Apoptosis wasdetermined in AR-specific and control siRNA plasmid transfected LNCaP cells. The transfected cells werethen treated with or without 3a-diol. A: LNCaP cells transfected with the pSiAR-EGFP plasmid wereincubated in their growth medium. B: pSiAR-EGFP transfected LNCaP cells were maintained in the growthmedium supplemented with 10�8 M 3a-diol. C: LNCaP cells transfected with nonspecific pSiCon-EGFPplasmid were cultured in the growth medium.

TABLE I. Quantification of 3a-Diol Effects on Apoptosis in AR-SilencedLNCaP Cells

Plasmid siRNA transfectionTUNEL positive

cells (% mean�SD)Transfectionefficiency (%)

pSiAR-EGFP 15.57� 1.15a,b 13.29pSiAR-EGFP treated with 3a-diol 7.10� 1.54a 13.50pSiCon-EGFP 4.11� 1.06 13.85

aThe percentages of TUNEL positive cells were significant higher in pSiAR-EGFP-transfected LNCaPcells, with or without 3a-diol supplementation, than in pSiCon-EGFP-transfected cells (P< 0.01).bThe percentages of TUNEL positive cells in pSiAR-EGFP-transfected LNCaP cells were significant higherthan their counterparts treated with 10�8 M 3a-diol (P< 0.01).

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activation would follow the temporal changesof 5a-DHT-activated responses. However, ourresults showed that 3a-diol-regulated Aktexpression peaks at 8 h whereas 5a-DHT-regulated Akt expression peaks at 30 min. Moresignificantly, 3a-diol activated more rapid androbust Akt phorphorylation as compared to 5a-DHT stimulation. 3a-diol-regulated Akt expres-sion and phosphorylation cannot be attributedto 5a-DHT formation, since 5a-DHT was notdetected in LNCaP cells following 3a-dioladministration. Such observations were consis-tent with results reported by Agapova et al.[2006] in which 3a-diol is shown to be morepotent than a synthetic 5a-DHT agonist instimulating PI3K/Akt signaling in optic nervehead astrocytes.

To evaluate the involvement of the AR in3a-diol-activated androgenic effects, AR expres-sion in LNCaP cells was suppressed usingsiRNA approaches. Despite the observationsthat the AR-specific siRNA duplex only tran-siently suppresses AR expression in LNCaPcells, the nearly completed suppression of ARexpression was appropriate for gene expressionand signal transduction studies. Levels of3a-diol-regulated b-catenin expression weresimilar between cells with and without the ARsuggesting that 3a-diol-regulated b-cateninexpression can be independent from the AR.Patterns of the 3a-diol-stimulated Akt expres-sion and phosphorylation were different in thepresence and the absence of the AR; and theresults might reflect a reciprocal interactionbetween the AR and PI3K/Akt signaling path-ways in LNCaP cells [Lin et al., 2003]. Theseresults, together with metabolism studies,suggest that 3a-diol-activated ARG expressionand cytoplasmic signaling can be independentof 5a-DHT and the AR. 3a-diol, therefore, mustactivate alternative pathways other than theAR to execute its androgenic actions.

Suppression of AR expression or disruptionof AR signaling has been shown to suppresscell proliferation [Chen et al., 1998; Eder et al.,2000; Wright et al., 2003] or to induce celldeath [Yang et al., 2005b]. Supplementationof 5a-DHT did not support AR-silenced LNCaPcell proliferation. This is consistent with theconcept that 5a-DHT utilizes AR pathway totransduce its androgenic signals. In contrast,3a-diol supported LNCaP cell proliferation in theabsence of the AR, and stimulated AR negativePC-3 cell proliferation. 3a-diol-stimulated PC-3

cell proliferation cannot be attributed to5a-DHT or the AR since 3a-diol is not oxidizedto 5a-DHT in PC-3 cells [Rizner et al., 2003] and5a-DHT suppresses cell proliferation in PC-3cells stably transfected with the AR [Yuanet al., 1993]. The 3a-diol-stimulated AR-silencedLNCaP cell proliferation might result fromreduced cell death. However, 3a-diol-activatedLNCaP cell survival pathways must work withserum components since supplementation of 3a-diol in a reduced serum conditioin (OPTI-MEMplus 2% CD FBS) did not support cell prolife-ration (data not shown). Consistent with otherreports that PI3K/Akt signaling is a dominantsurvival pathway in LNCaP cells [Lin et al.,1999; Sun et al., 2003; Yang et al., 2005a], theinclusion of a PI3K inhibitor, LY294002, sup-pressed cell survival in both 3a-diol and 5a-DHT-treated LNCaP cells (data not shown).Although it is still unclear which mechanismthat 3a-diol uses to rescue cell death andpromote cell proliferation, 3a-diol-activated,AR-independent prostate cell survival and pro-liferation may result from Akt and/or b-cateninactivation (Fig. 7), since elevated PI3K/Aktsignaling through a variety of stimuli cancontribute to the failure of androgen depriva-tion therapy [Murillo et al., 2001; Lin et al.,2003].

Testosterone is synthesized from the testesand the adrenal glands, and converted torelated androgen metabolites in the prostateusing tissue-specific steroidogenic enzymes.Androgen deprivation therapy is intended toblock potent androgen accumulations. How-ever, levels of potent androgens remain rela-tively constant in the prostate before and afterthe therapy [Nishiyama et al., 2004; Tituset al., 2005]. Abnormal intraprostatic androgenmetabolism and androgen metabolite accumu-lation can result from deregulated androgenmetabolizing enzyme expression [Mizokamiet al., 2004]. There are two major 3a-HSDisozymes, AKR1C2 and AKR1C3, in the humanprostate [Lin et al., 1997; Penning et al., 2000].Both isozymes catalyze mainly 5a-DHT reduc-tion for 3a-diol formation [Lin et al., 1997;Rizner et al., 2003]. Elevated expression of3a-HSDs has been observed in primary culturesof human prostate cancer cells as compared tothose derived from normal prostate [Lin et al.,1997; Rizner et al., 2003], and confirmed inlocalized and advanced prostate cancer tissues[Nakamura et al., 2005; Fung et al., 2006;

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Stanbrough et al., 2006]. Based on this study,levels of intraprostatic 3a-diol need to bedetermined in order to establish roles of 3a-HSDs and 3a-diol in prostate cancer develop-ment and progression. Future therapeuticdevelopment for treating advanced prostatecancer might need to target reductive 3a-HSDsto suppress intraprostatic accumulation of 3a-diol in conjunction to current androgen depri-vation therapy.

ACKNOWLEDGMENTS

This work was supported by the US Depart-ment of Veterans Affairs Merit Award to HKL.

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