doxazosin induces apoptosis of benign and malignant...

Doxazosin Induces Apoptosis of Benign and Malignant Prostate

Cells via a Death Receptor–Mediated Pathway

Jason B. Garrison and Natasha Kyprianou

Department of Molecular and Cellular Biochemistry and Division of Urology, Department of Surgery, University of KentuckyCollege of Medicine, Lexington, Kentucky

Abstract

Quinazoline-based A1-adrenoceptor antagonists such as dox-azosin and terazosin have been previously shown to induceapoptosis in prostate cancer cells via an A1-adrenoceptor–independent pathway, involving activation of transforminggrowth factor-B1 (TGF-B1) signaling. In this study, themolecular events initiating this apoptotic effect were furtherinvestigated in vitro using the human androgen-independentprostate cancer cells PC-3 and the human benign prostateepithelial cells BPH-1. Quantitative microarray assays weredone in PC-3 and BPH-1 cells after treatment with doxazosin(25 Mmol/L, 6 and 24 hours) to identify the early gene changes.Transient changes in the expression of several apoptosisregulators were identified, including up-regulation of Bax andFas/CD95 and down-regulation of Bcl-xL and TRAMP/Apo3.Moreover, there were significant changes in the expressionpattern of signaling components of the extracellular matrixsuch as integrins A2, AV, B1, and B8. Western blot analysisrevealed activation of caspase-8 and caspase-3 within the first6 to 12 hours of treatment with doxazosin in both PC-3 andBPH-1 cells. Doxazosin-induced apoptosis was blocked byspecific caspase-8 inhibitors, supporting the functional in-volvement of caspase-8 in doxazosin-induced apoptosis. Theeffect of doxazosin on recruitment of Fas-associated deathdomain (FADD) and procaspase-8 to the Fas receptor wasexamined via analysis of death-inducing signaling complexformation. Doxazosin increased FADD recruitment andsubsequent caspase-8 activation, implicating Fas-mediatedapoptosis as the underlying mechanism of the effect ofdoxazosin in prostate cells. These results show that doxazosinexerts its apoptotic effects against benign and malignantprostate cells via a death receptor–mediated mechanism witha potential integrin contribution towards cell survival out-comes. (Cancer Res 2006; 66(1): 464-72)

Introduction

Dysfunctional apoptotic programming leading to loss/suppres-sion of apoptosis has been causally implicated in prostate cancerdevelopment and progression; thus, the apoptotic process becomesa promising therapeutic target for the effective elimination ofprostate cancer cells (1). The two major apoptotic pathways are themitochondrial dependent pathway and the death receptor pathway(2). In response to specific signals (such as DNA damage), the

mitochondrial pathway occurs via the release of cytochrome c andformation of the apoptosome, a multiprotein complex containingApaf-1, cytochrome c , and caspase-9. Activation of caspase-9triggers a cascade of effector caspases and subsequent apoptoticcell death. The receptor-mediated pathway is activated at the cellmembrane by receptors such as Fas/CD95 and TRAIL/Apo3, whichon ligand binding initiate recruitment of the tumor necrosis factor(TNF) receptor–associated death domain (TRADD), Fas-associateddeath domain (FADD), and procaspase-8 to form the death-inducing signaling complex (DISC). The Fas/Fas ligand signalingpathway has been implicated in the progression and chemo-prevention of prostate cancer (3–5). Fas can be activated bymolecules besides Fas ligand or anti-Fas antibody, and variouschemotherapeutic agents have been shown to enhance apoptosis ofandrogen-independent human prostate cancer cells via activationof the Fas signaling pathway (6–8). Activation of the caspasecascade mediates apoptosis in prostate cancer cells in response toa number of cytotoxic agents as well as transforming growth factorh (TGF-h; refs. 9–11).a1-Adrenoreceptor antagonists, such as quinazoline-based com-

pounds doxazosin and terazosin, are clinically effective in the reliefof benign prostatic hyperplasia (BPH) symptoms via their ability toselectively antagonize the a1a-adrenoreceptors (12). Recent exper-imental and clinical evidence, however, indicates that induction ofprostate epithelial and smooth muscle cell apoptosis by doxazosinand terazosin is one of the molecular mechanisms contributingto the overall long-term clinical efficacy of these medications inimproving lower urinary tract symptoms in BPH patients (13).Suppression of prostate growth proceeds via an a1-adrenoceptor-independent mechanism by activation of latent apoptotic machin-ery by effector (Smad) activation of TGF-h1 signaling and InBa(14, 15). More recent evidence established the ability of doxazosinto induce anoikis in prostate epithelial and endothelial cells (16).Additional signaling mechanisms involving disruption of cellattachment to the extracellular matrix (ECM) and subsequentinduction of anoikis have been functionally implicated (16) in amolecular cross-talk of the cell death actions of quinazolines; themolecular signaling of this apoptotic effect, however, has yet tobe defined.In this study, we hypothesize that doxazosin induces prostate

cancer cell apoptosis by affecting cell migration/anoikis via the Fasreceptor pathway. Our results, based on cDNA array analysis andtemporal dissection of the events leading to the apoptotic outcomeof benign and malignant human prostate cells, show the ability ofdoxazosin to activate the Fas-mediated apoptotic pathway.

Materials and Methods

Cells Lines and TransfectionsThe human androgen-independent prostate cancer cell line PC-3 was

obtained from the American Type Tissue Culture Collection (Rockville, MD).

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

Requests for reprints: Natasha Kyprianou, Division of Urology, MS-283, Universityof Kentucky Medical Center, 800 Rose Street, Lexington, KY 40536. Phone: 859-323-9812; Fax: 859-323-1944; E-mail: [email protected].

I2006 American Association for Cancer Research.doi:10.1158/0008-5472.CAN-05-2039

Cancer Res 2006; 66: (1). January 1, 2006 464 www.aacrjournals.org

Research Article

Research. on July 28, 2019. © 2006 American Association for Cancercancerres.aacrjournals.org Downloaded from

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PC-3 cells were routinely cultured in RPMI 1640 purchased from Invitrogen(Carlsbad, CA) containing 10% fetal bovine serum (Invitrogen) and

antibiotics. The nontumorigenic benign human prostatic epithelial cells

BPH-1 (derived from human prostate epithelium of benign pathology; a

generous gift from Dr. Simon W. Hayward, Department of UrologicalSurgery, Vanderbilt University Medical Center, Nashville, TN; ref. 17) were

cultured in RPMI 1640 (Invitrogen) containing 10% fetal bovine serum and

antibiotics.

Human prostate cancer cells PC-3 were transfected with a dominant-negative form of FADD (DN-FADD) made with PCR primers (5V-CCAAGCT-TATGGACGACTTCGAGGCGGGG and 5V-AAGGATCCACCAGCGCAAAG-CAGCGGCC) flanking the region encoding 117 to 208 amino acids and

using the FADD cDNA as template. This region contains the death domainthat binds to the clustered receptor death domain but does not contain the

death-effector domain and therefore cannot bind procaspase-8. The HindIII/

BamHI restriction sites were used for cloning DN-FADD into the expressionvector pcDNA3.1/Hygro. Empty vector was used to obtain control cells

resistant to hygromycin.

Wild-type (WT)-FADD was cloned into a modified pcDNA3 expression

vector (Invitrogen) in which a hemagglutinin (HA) epitope tag (YPYDVPDYA)had previously been placed downstream of the cytomegalovirus promoter/

enhancer (pcDNA3 HA-FADD). In addition, an AU1 epitope (DTYRYI)-tagged

FADD and mutants were made with PCR primers encoding the epitope and

using the FADD cDNA as template (pcDNA3 AUl-FADD). Empty vector wasused to obtain control cells resistant to G418. FuGENE6 reagent kit (Roche,

Basel, Switzerland) was used for transfection and cells were selected in the

presence of hygromycin (DN-FADD) or G418 (WT-FADD).

ReagentsDoxazosinmesylate (Cardura) was a gift from Pfizer Pharmaceuticals (New

York, NY). Collagen type I was purchased from Sigma-Aldrich Co.

(St. Louis, MO). Fibronectin and z-IETDfmk caspase-8 inhibitor were pur-

chased form BD Biosciences (Bedford, MA). Recombinant human vascular

endothelial growth factor was purchased from R&D Systems, Inc. (Minneap-

olis, MN). The polyclonal anti-Bax, monoclonal anti–caspase-3 and caspase-8,

and polyclonal anti-FADD were purchased from Cell Signaling Technology

(Beverly, MA). The monoclonal anti-TRADD was purchased from BD Bio-

sciences. The polyclonal anti-c-FLIPL was purchased from Chemicon Inter-

national (Temecula, CA). The monoclonal anti-Fas antibody was purchased

from StressGen Biotechnologies Corp. (Victoria, British Columbia, Canada).

RNA Isolation and AnalysisTotal RNA was extracted from treated and untreated prostate cells by

TRIzol (Invitrogen). cDNA was labeled from total RNA (4 Ag) with biotin-16-

dUTP and the GEArrayTM AmpoLabelling-LPR Kit (SuperArray, Frederick,

MD) based on the protocol of the manufacturer. The labeled cDNA was then

hybridized overnight to GEArray Q Series Human Cancer PathwayFinder,

GEArray Q Series Human ECM and Adhesion Molecule, GEArray Q Series

Human Apoptosis, or GEArray Q Series Human NFnB Signaling Pathway

gene arrays (SuperArray) according to the protocol of the manufacturer.

Signal detection was achieved by exposure to CDP-Star alkaline phospha-

tase chemiluminescent substrate (SuperArray).

Data analysis was done using a UVP Bioimaging System (Upland, CA)which converted the images into numerical data. Raw signal intensities were

corrected for background by subtracting the signal intensity of the average

Figure 1. Effect of doxazosin on cell viability andapoptosis of malignant and benign prostate epithelial cells.Subconfluent cultures of PC-3 and BPH-1 cells wereexposed to increasing concentrations of doxazosin(0-35 Amol/L) and cell death was determined using theMTT assay (A) or stained with Hoechst (B ); apoptoticcells were visualized and counted as described inMaterials and Methods. Points, mean of threeindependent experiments done in duplicate; bars, SE.*, P < 0.01; **, P < 0.001, compared with the control.

Fas-Mediated Apoptosis by Doxazosin

www.aacrjournals.org 465 Cancer Res 2006; 66: (1). January 1, 2006



between two sets of housekeeping genes, h-actin and glyceraldehyde-3-phosphate dehydrogenase, or a blank. The normalized signals were used to

determine the relative fold change of particular transcripts before and after

treatment.

Apoptosis and Cell Viability Assay EvaluationHoechst staining. Cells were plated on six-well plates at 5 � 104 per

well and at subconfluency were treated with increasing concentrations of

doxazosin (1, 10, and 25 Amol/L) in the presence or absence of the caspase-

8 inhibitor z-IETDfmk. After 24 and 48 hours of treatment, cells were fixedwith 4% (w/v) paraformaldehyde (Sigma) and stained with 10 Ag/mL

Hoechst 33342 (B2261; Sigma) in the presence of 0.1% Triton X-100 (Sigma)

as previously described (1). Cells were visualized using a Zeiss Axiovert S100

fluorescent microscope (Thornwood, NY) with a UV filter (365 nm) andthose cells containing fragmented nuclei were designated apoptotic (�100

magnification). The apoptotic index was determined by counting three

random fields in duplicate wells per treatment group. Each experiment was

done in two independent experiments.Trypan blue exclusion assay. Subconfluent cultures of cells were

exposed to increasing concentrations of doxazosin (0-25 Amol/L) and cell

viability was determined after 24 hours via the trypan blue exclusion assay

(14). Values from three independent experiments (done in duplicate) areexpressed as mean percent of cell viability relative to untreated cultures.

3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay.Subconfluent cultures of cells were exposed to increasing concentrations ofdoxazosin (0-35 Amol/L). After treatment, the medium was replaced with

250 AL of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide

(MTT; 1 mg/mL; Sigma) and incubated at 37jC to form blue crystals. After

2 hours, the MTT was removed and replaced with 250 AL DMSO andincubated overnight at 37jC. The absorbance of the DMSO-crystal solution

was read at 540 nm in a microplate reader (Bio-Tek Instruments, Winooski,

VM). The percent change in cell viability was determined by comparing the

absorbance values. Numerical data represent the average of two independentexperiments done in triplicate.

Measurement of Caspase-8 ActivityThe caspase-8 activity was determined using the colorimetric assay kit

(Chemicon International) as described by the protocol of the manufac-

turer. Cultures of malignant (PC-3) and benign (BPH-1) prostate epithelialcells (2 � 106), treated at subconfluency with doxazosin (25 Amol/L)

for 24 and 48 hours, were lysed in cell lysis buffer and equivalent amounts

of protein in each sample were incubated with the assay mixture

containing IETD-pNA for 2 hours at 37jC. The absorbance was read at405 nm in a microplate reader (Bio-Tek Instruments). Fold increase in

activity was determined by comparing the absorbance values. Numerical

data represent the average of two independent experiments done in

duplicate.

Western Blot AnalysisCultures of PC-3 and BPH-1 cells treated with doxazosin (25 Amol/L)

for 0, 6, 12, or 24 hours were lysed in radioimmunoprecipitation assay

(RIPA) buffer [150 mmol/L NaCl, 50 mmol/L Tris (pH 8.0), 0.5%

deoxycholic acid, 1% NP40 with 1 mmol/L phenyl methyl-sulfonylfluoride]. The total protein concentration in each sample was quantified

by bicinchoninic acid (BCA) protein assay kit (Pierce, Rockford, IL) and

protein samples (30 Ag) were subjected to SDS-PAGE and transferred to

Hybond-C membranes (Amersham Pharmacia Biotech, Piscataway, NJ).Membranes were blocked with 5% dry milk in TBS-T (TBS containing

0.05% Tween 20) for 1 hour at room temperature and were subsequently

incubated overnight at 4jC with antibodies against caspase-3 or

caspase-8, Akt, or phosphorylated Akt (Cell Signaling Technology).After incubation with the respective primary antibody, membranes were

exposed to species-specific horseradish peroxidase–labeled secondary

antibodies (room temperature). Signal detection was achieved withSuperSignal West Dura Extended Duration Substrate (Pierce) and

visualized using a UVP Imaging System. All bands were normalized to

a-actin expression (Oncogene Research Products, La Jolla, CA) and fold

changes in protein expression were determined on the basis of a-actinloading control.

ImmunoprecipitationBenign and malignant prostate epithelial cells were treated with

doxazosin (25 Amol/L) for 0, 6, 12, or 24 hours and were subsequently

Table 1. Gene expression in PC-3 and BPH-1 cells inresponse to doxazosin (6 hours)

Genebank Gene name Fold change

PC-3 BPH-1

Apoptosis related

NM_001160-1 Apaf-1 1.3 �2.3

L22474 Bax 11.8 3.0Z23115 Bcl-x 13.8 ND

NM_004346 caspase-3 �1.6 ND

NM_001228 caspase-8 �2.5 2.0NM_004049 FADD 3.4 ND

X63717 Fas/CD95 4.3 3.2

U74611 DR3/Apo3 �1.3 ND

NM_002392 Mdm2 �2.1 �1.5NM_003894 RIP �2.8 ND

U75285 survivin ND �1.7

NM_000594 TNFa 1.7 ND

NM_001065 TNFR1 ND �1.7NM_005658 TRAF1 3.3 ND

NM_004620 TRAF6 1.1 �3.3

NOTE: ND, not detected.

Table 2. Effect of doxazosin on ECM/adhesion geneexpression in PC-3 prostate cancer cells

Genebank Gene name Fold change

6 h 24 h

Z13009 E-cadherin 1.8 �1.5

NM_004363 CEA �13.7 �2.3NM_001904 catenin b1 3.5 1.3

AF062343 catenin d1 �1.0 �1.2

X02761 fibronectin-1 �2.8 �1.6

X17033 integrin a2 2.3 2.3M59911 integrin a3 �2.1 �2.8

X06256 integrin a5 �2.5 �2.5

NM_002210 integrin aV �3.2 �3.7

NM_002211 integrin b1 �2.6 �2.4X53587 integrin b4 �4.2 �5.1

J05633 integrin b5 �2.3 �3.8

NM_000888 integrin b6 �3.4 �3.2NM_002214 integrin b8 1.1 2.4

M61916 laminin b1 �1.7 �2.0

J03202 laminin b2 1.8 �2.6

NM_000247 MUC-18 1.2 2.2M83248 osteopontin 7.3 6.4

NM_000442 PECAM-1 �2.6 �1.7

M30640 E-selectin 2.4 �2.3

NM_003006 P-selectin 1.1 1.2NM_003246 TSP-1 �1.4 �1.2

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lysed in RIPA buffer. Total protein concentration was quantified by BCA

protein assay kit (Pierce) and protein samples (30 Ag) were incubated

overnight at 4jC with the specific antibody. Lysates were then incubated for

3 hours with Protein G Plus/Protein A agarose beads (30 AL; Oncogene).Beads were washed five times with RIPA buffer, subjected to SDS-PAGE, and

blot transferred to nitrocellulose membranes (Amersham Pharmacia

Biotech).

Statistical AnalysisOne-way ANOVA was done using the StatView statistical program to

determine the statistical significance between values. All data are presented

as mean F SE. P < 0.05 was considered statistically significant.

Results

Doxazosin causes a loss of cell viability and inducesapoptosis in malignant and benign prostate epithelial cells.Doxazosin leads to a dose-dependent loss of PC-3 (14) and BPH-1cell viability after 24 hours of treatment as determined by MTTassay (Fig. 1A). A similar profile of loss of cell viability was obtainedusing the trypan blue exclusion assay (data not shown). Theapoptotic nature of this effect was documented using the Hoechststain in benign prostate epithelial cells exposed to doxazosin. Asshown in Fig. 1B , there was a significant increase in the numberof apoptotic cells at 24 and 48 hours of exposure to the drug(as identified by fragmented nuclei).

Doxazosin-induced gene expression profile of BPH-1 andPC-3 cells. In an attempt to identify the genes differentiallyexpressed in response to doxazosin in benign and malignantprostate epithelial cells were, a gene expression array analysis wasdone after treatment of cells with doxazosin (25 Amol/L). Table 1summarizes the data of the gene transcription profile of genesknown to play a critical role in apoptosis. After a 6-hour treatmentwith doxazosin, several genes were significantly up-regulatedin PC-3 cells: Bax, Bcl-xL, FADD, and Fas. No detectable levels ofFADD mRNA were observed in BPH-1 cells whereas Fas mRNAwas up-regulated in response to doxazosin treatment. The geneexpression pattern was different between the two cell lines inresponse to doxazosin (Table 1).Considering that doxazosin might elicit a potential anoikis effect

(6), we did an array analysis of genes functionally involved in celladhesion, migration, and ECM attachment on PC-3 cells. Theresults summarized in Table 2 revealed a considerable reduction inthe levels of mRNA corresponding to integrins a3, a5, aV, h1, h4, h5,and h6 whereas there was an increase in mRNA levels for integrinsa2 and h8. Also shown in Table 2 are data indicating alterations ingene expression for several molecules involved in cell adhesion.Major changes were detected for E-cadherin, carcinoembryonicantigen, h-cantenin, platelet/endothelial cell adhesion molecule 1(PECAM-1), laminins, selectins, and thrombospondin-1 (TSP-1).

Figure 2. Doxazosin causes an increase in Baxprotein and induces caspase-8 activation in benignand malignant prostate epithelial cells. Westernblot analysis was applied to cell lysates fromdoxazosin-treated PC-3 and BPH-1 cells as describedin Materials and Methods. A, following treatment, PC-3and BPH-1 cells have an increase in Bax proteinexpression. B, PC-3 and BPH-1 cells show cleavageof procaspase-8 into its active form. PC-3 cellsshow increased levels of FADD protein. No changes inFLIP levels were observed in either cell line.C, both PC-3 and BPH-1 cells show a significantincrease in caspase-8 activity at 24 and 48 hours.*, P V 0.02.





Driven by the findings indicating that doxazosin up-regulatesthe mRNA of the proapoptotic Bax, we subsequently investigatedthe ability of doxazosin to regulate additional key apoptosis playersof the mitochondrial pathway. Western blot analysis revealed a2.5- and 3-fold increase in Bax protein levels in response todoxazosin in PC-3 cells at 12 and 24 hours, respectively. Further-more, in BPH-1 cells there was a 2.5-fold increase in Bax after24 hours of doxazosin treatment compared with untreated cells(Fig. 2A). This temporally correlates with the observed changes inmRNA expression.Caspase-8 activation by doxazosin. Western blotting indicates

an increase in the levels of activated caspase-8 at 6 hours followingdoxazosin treatment in BPH-1 cells (Fig. 2B). For the PC-3 cells,this caspase activation was observed after 12 hours (Fig. 2B). After6 hours of treatment, PC-3 cells show an increase in FADD proteinlevels whereas BPH-1 cells show no change, consistent with ourgene array data (Fig. 2B). There were no major changes in c-FLIPLprotein expression in prostate cell lines. Furthermore, on stimu-lation with doxazosin (25 Amol/L), both PC-3 and BPH-1 cellsexhibited a significant increase in caspase-8 activity compared withcontrol (Fig. 2C).The data shown in Fig. 3 indicate that there was a significant

suppression of apoptosis in the presence of the caspase-8 inhibitorz-IETDfmk in both the benign and malignant prostate cells.Doxazosin induces caspase-3 activation and death receptor

and adaptor protein complex formation. We have previouslyreported that doxazosin treatment of malignant prostate cellscauses activation of caspase-3 (15). To further dissect the molecularpathway of apoptosis triggered by doxazosin in benign prostateepithelial cells, caspase-3 activation was determined by Westernblot analysis. Following caspase-8 activation, cleavage of full-lengthcaspase-3 was seen in BPH-1 cells after 12 hours of doxazosintreatment (Fig. 4A). Interestingly, there was no change in TRADDprotein expression following treatment with doxazosin (data notshown).To determine if caspase-8 activation was a result of Fas

activation and/or increased FADD recruitment, their complexformation was analyzed. Immunoprecipitation analysis showed anincrease in FADD and caspase-8 interactions in both PC-3 andBPH-1 cell lines following treatment with doxazosin (Fig. 4B).Moreover, there was an increased association of FADD and Fasfollowing treatment of both cell lines (Fig. 4B).Doxazosin causes FADD-dependent apoptosis. Prostate can-

cer cells PC-3 were stably transfected with either a WT-FADD or aDN-FADD. High levels of WT-FADD and DN-FADD were detected inthe PC-3 transfectants by Western blot analysis (data not shown).Four clones from each PC-3 WT-FADD and DN-FADD were selectedto determine the effects doxazosin on cell viability. Doxazosintreatment results in a dose-dependent decrease in cell viability inPC-3 WT-FADD transfectants (Fig. 5A). In contrast, there were nosignificant changes in cell death of PC-3 DN-FADD transfectantscompared with parental cells in response to doxazosin (Fig. 5B). Asshown in Fig. 5C , overexpression of WT-FADD in prostate cancercells resulted in a significant increase in doxazosin-inducedapoptosis in the four cloned transfectants whereas PC-3 cellsexpressing the DN-FADD exhibited a reduced apoptotic response todoxazosin (Fig. 5C and Supplementary Fig. S1).Doxazosin causes a decrease in Akt phosphorylation. We

recently reported that doxazosin-induced apoptosis in prostatecancer cells was mediated via inhibition of intracellular Aktactivation by targeting its phosphorylation (18). Figure 6 shows a

decrease in Akt phosphorylation with no apparent change in Aktprotein levels in response to doxazosin (25 Amol/L) in PC-3 cellsfollowing normalization to actin. In contrast, PC-3 DN-FADDtransfected cells show no change in the levels of phosphorylatedAkt in response to doxazosin as determined by densitometricanalysis of total Akt/pAkt.

Discussion

Apoptosis targeting coincides with the goal of successfullytreating patients with advanced prostate cancer. A large number ofhuman tumors exhibit down-regulation of, or insensitivity to,

Figure 3. Suppression of doxazosin-induced apoptosis by caspase-8 inhibitor.Benign (BPH-1) and malignant (PC-3) prostate cells were treated withdoxazosin (25 Amol/L) in the presence or absence of the specificcaspase-8 inhibitor z-IETDfmk (20 Amol/L). Apoptosis induction was evaluatedafter 24 and 48 hours as described in Materials and Methods. PC-3 (A)and BPH-1 (B ) cells showed a significant decrease in the number of apoptoticcells. *, P < 0.05.

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proapoptotic molecules such as Fas and FADD or up-regulationof antiapoptotic molecules like FLIP, suggesting that these cellspossess regulatory mechanisms blocking the death signal receivedthrough the death receptors (19–21).Induction of apoptosis in response to doxazosin is well

documented in benign and malignant prostate epithelial cellsand human prostate clinical specimens at intracellular concen-trations comparable with the therapeutic doses (13). The presentdata indicate that doxazosin also causes a significant loss ofcell viability and enhanced apoptosis of benign prostate cellsin vitro. Interestingly enough, benign prostate cells seem to bemore sensitive to the apoptotic effect of doxazosin than tumorcells (16).The data from the gene array analysis revealed that a modest

change in down-regulation in caspase-8 mRNA in both cells linesafter treatment is in agreement with recent evidence indicating nomajor changes in the mRNA levels of caspase-8 during the periodpreceding the onset of apoptosis (22). Doxazosin treatment ofbenign and malignant prostate cells resulted in cleavage andactivation of caspase-8, and caspase activation parallels thedecreased cell viability in a time-dependent manner, indicative ofa Fas-receptor pathway for apoptosis induction. In a similartemporal pattern to malignant prostate epithelial cells (15), benignprostate cells exhibited cleavage of caspase-3 after 12 hours oftreatment.Caspase-8 activation is preceded by its association with FADD

and their subsequent recruitment by Fas. In addition, all humanprostate cancer cell lines express Fas, but most are resistant toapoptosis induced by an agonistic anti-Fas antibody (23). SomeFas-resistant cell lines will go through apoptosis by concurrentincubation with a Fas antibody and a protein synthesis inhibitoror by sensitizing the cells with chemotherapeutics (24, 25). Thiswould support the notion that some chemotherapeutic agentscan sensitize tumor cells to Fas-mediated apoptosis (25, 26). Anumber of anticancer drugs activate the death receptor pathwayvia enhancement of Fas and Fas ligand expression (27). Wefound an increase in both Fas mRNA and protein levels with nochange in Fas ligand mRNA expression in either cell line aftertreatment with doxazosin, consistent with reported evidence that

Fas ligand is not required for drug-induced apoptosis becauseapoptosis is not suppressed by inhibition of Fas/Fas ligandinteraction (27, 28).One could argue that FADD expression may contribute to the

sensitivity of Fas activation in prostate cancer cells, as shown intongue carcinoma cells in response to carboplatin (29). Moreover,an increase in FADD and procaspase-8 expression has been shownin colon carcinoma cells on treatment with cisplatin, doxorubicin,or mitomycin C (30, 31). In prostate cancer cells, we show anincrease in FADD mRNA and protein expression as well as anincreased DISC formation in response to doxazosin. In agreementwith our data, other groups have shown similar increases inFADD and DISC formation in tumor cells following drugtreatment (32).FADD protein has been shown to interact with caspase-8 via

its death-effector domain and blocking proteolytic activation ofcaspase-8 at the DISC prevents apoptosis (33). Considering thisevidence, we examined the functional consequences of WT-FADDoverexpression and the expression of DN-FADD (lacking death-effector domain) on doxazosin-induced apoptosis of prostate cells.PC-3 cells overexpressing WT-FADD exhibited a dramatic increasein their apoptotic response to doxazosin whereas cells expressingDN-FADD have a significant decrease in doxazosin-mediatedapoptosis. This further confirms our hypothesis that doxazosin-induced apoptosis occurs via increased FADD protein levels andDISC formation.The role of Akt signaling in regulating death receptor signaling

is not understood. We have previously shown that exposureto doxazosin causes a decrease in Akt phosphorylation (18).Mechanistically, Akt activation promotes the degradation of InB,which is involved in the apoptotic action of doxazosin inprostate cancer cells (15). In other cellular systems, such as Tlymphocytes, blocking Akt signaling has been shown to increasecaspase-8 activity, ultimately resulting in Fas-dependent apopto-sis (34). Moreover, canstatin inhibits Akt activation and inducesFas-dependent apoptosis in endothelial cells (35). Taken together,this evidence strongly supports Akt signaling as a criticaldownstream target of the death receptor–mediated apoptoticpathway.

Figure 4. Effect of doxazosin on caspase-3 activation and DISC formation. A, BPH-1 cells were treated with doxazosin (25 Amol/L) for the time periodsindicated and cell lysates (30 Ag of protein) were subjected to Western blotting. The antibody showed activation of caspase-3 at 12 and 24 hours. B, benign andmalignant prostate epithelial cells were exposed to doxazosin (25 Amol/L) for 6, 12, and 24 hours and DISC formation was determined using immunoprecipitationassays. Specific antibodies and conditions are described in Materials and Methods. Following treatment, PC-3 and BPH-1 cells show increased association betweenmolecules involved in the DISC.





A mutant form of FADD would potentially uncouple caspasesignaling and block apoptosis. Indeed, the present data indicateno changes in Akt activation in doxazosin-treated DN-FADDclones correlating with the apoptotic response compared withthe untreated controls. A similar pattern has been found inanother experimental system of apoptosis in staurosporinetreated cells expressing a DN-FADD (36). Moreover, apoptosisinduction and detachment from ECM can be blocked byexpression of a DN-FADD mutant with concurrent treatmentwith various chemotherapeutic agents (31, 37, 38). Studies byother investigators have also shown that cisplatin and campto-thecin induce Fas ligand–independent aggregation of death

receptors and recruitment of FADD to death receptors (37).Further experiments are required to examine the possible cross-talk between Akt and FADD.In endothelial cells, Fas/Fas ligand interaction, Fas-FADD

complex formation, and caspase-8 activation have been implicatedas the precursor to anoikis, and inhibition of any of these eventsblocks anoikis (39). As shown in the present study, doxazosin-mediated apoptosis may also rely on changes in integrinexpression at the cell surface that may cause anoikis. Consideringthat anoikis-resistant cells are able to detach from the primarytumor without undergoing apoptosis, acquisition of anoikisresistance becomes a critical step in the metastatic process.

Figure 5. Effect of doxazosin on cellviability and apoptosis of PC-3 WT-FADDand DN-FADD transfectants. Subconfluentcultures of PC-3 transfectant clone cellswere exposed to increasing concentrationsof doxazosin (0-35 Amol/L) and cell deathwas determined using the MTT assay.A, following exposure, PC-3 WT-FADDshows a decreased number of viable cellsat 24 and 48 hours. B, PC-3 DN-FADDshows no change in cell viability followingtreatment for 24 and 48 hours. Points,mean percentage of cell viability fromthree independent experiments done induplicate; bars, SE. C, Following treatmentwith doxazosin, cells were stained withHoechst and apoptotic cells were visualizedand counted as described in Materials andMethods. Columns mean of threeindependent experiments done induplicate; bars, SE.

Cancer Research




Overexpression of a number of mediators involved in cell-matrixand cell-cell anchorage can provide resistance to anoikis, as well asthe tumor suppressor genes PTEN and p53 , or overexpression ofoncogenes such as ras, raf , and src (40). The Fas-receptor signalingpathway and bcl-2 overexpression have also been intimatelyimplicated in anoikis (37, 41).In summary, the present study indicates that doxazosin

treatment of benign and malignant prostate epithelial cells leadsto a significant increase in DISC formation and subsequentapoptosis via caspase-3 activation. The apoptotic effect ofdoxazosin provides a molecular basis for therapeutic targeting ofprostate cancer, as well as benign disease, potentially via anoikis.Based on the present findings, a model can be proposed featuringcell detachment as the critical event in apoptosis induction bydoxazosin, potentially with caspase-3 activation as a consequenceof loss of critical cell attach-ments rather than the contributing

executioner of apoptotic cell death. Ongoing studies address theinvolvement of integrin signaling in apoptosis induction in prostatetumor epithelial and endothelial cells and the development of novelantiangiogenic (quinazoline-based analogs) against androgen-independent prostate tumors.

Acknowledgments

Received 6/15/2005; revised 10/7/2005; accepted 10/21/2005.Grant support: NIH grant R01 CA107575-01.The costs of publication of this article were defrayed in part by the payment of page

charges. This article must therefore be hereby marked advertisement in accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.

We thank Dr. Michael Cohen, Department of Pathology, University of Iowa, forproviding the FADD dominant-negative construct (DN-FADD, ref. 42), Dr. Vishva M.Dixit, Department of Pharmaceutical Chemistry, University of California, SanFrancisco, for providing the full-length FADD construct (43), James V. Partin forexpert assistance with the microarray data analysis, and Lorie Howard for help withthe submission of the manuscript.

Figure 6. Reduction in Akt phosphorylation by doxazosin.Western blot analysis was applied to cell lysates fromdoxazosin-treated PC-3 and PC-3 DN-FADD1 cells asdescribed in Materials and Methods. Following treatment,PC-3 cells show a decrease in Akt phosphorylationwhereas PC-3 DN-FADD1 cells show no change.

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Cancer Research




2006;66:464-472. Cancer Res Jason B. Garrison and Natasha Kyprianou

Mediated Pathway−Prostate Cells via a Death Receptor Doxazosin Induces Apoptosis of Benign and Malignant

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