chemoprevention of prostate cancer by d l-sulforaphane is … · chemoprevention of prostate cancer...

Prevention and Epidemiology

Chemoprevention of Prostate Cancer by D,L-Sulforaphane IsAugmented by Pharmacological Inhibition of Autophagy

Avani R. Vyas1, Eun-Ryeong Hahm1, Julie A. Arlotti2, Simon Watkins3, Donna Beer Stolz3,Dhimant Desai4, Shantu Amin4, and Shivendra V. Singh1,2

AbstractThere is a preclinical evidence that the oral administration of D,L-sulforaphane (SFN) can decrease the

incidence or burden of early-stage prostate cancer [prostatic intraepithelial neoplasia (PIN)] and well-differen-tiated cancer (WDC) but not late-stage poorly differentiated cancer (PDC). Because SFN treatment inducescytoprotective autophagy in cultured human prostate cancer cells, the present study tested the hypothesis thatchemopreventive efficacy of SFN could be augmented by the pharmacologic inhibition of autophagy usingchloroquine (CQ). Incidence of PDC characterized by prostate weight of more than 1 g was significantly lower inthe SFN þ CQ group than in control (P ¼ 0.004), CQ group (P ¼ 0.026), or SFN group (P ¼ 0.002 by Fisher exacttest). Average size of themetastatic lymph nodewas lower by about 42% in the SFNþCQ group than in control (P¼ 0.043 by Wilcoxon test). On the other hand, the SFN þ CQ combination was not superior to SFN alone withrespect to inhibition of incidence or burden of microscopic PIN orWDC. SFN treatment caused in vivo autophagyas evidenced by transmission electron microscopy. Mechanistic studies showed that prevention of prostatecancer and metastasis by the SFNþ CQ combination was associated with decreased cell proliferation, increasedapoptosis, alterations in protein levels of autophagy regulators Atg5 and phospho-mTOR, and suppression ofbiochemical features of epithelial–mesenchymal transition. Plasma proteomics identified protein expressionsignature that may serve as biomarker of SFN þ CQ exposure/response. This study offers a novel combinationregimen for future clinical investigations for prevention of prostate cancer in humans.Cancer Res; 73(19); 5985–95.�2013 AACR.

IntroductionCruciferous vegetable intake in the diet is inversely asso-

ciated with the risk of different malignancies including cancerof the prostate (1, 2), which is a leading cause of cancer-relateddeaths among men in the United States (3). Phytochemicalscapable of eliciting cancer protective effect have now beenisolated from different edible cruciferous vegetables includ-ing broccoli, watercress, and garden cress (4, 5). Sulforaphane[1-isothiocyanato-4-(methylsulfinyl)-butane] is one suchsmall molecule of interest for prevention of cancers (5). Sulfor-aphane (SFN) occurs naturally as an L-isomer, but its syntheticD,L-analogue has been studied extensively for cancer preven-tion properties (5–10). Naturally occurring or synthetic SFN

has exhibited cancer chemopreventive effects in chemicallyinduced as well as oncogene-driven rodent cancer models(6–10). Talalay and colleagues were the first to report SFN-mediated inhibition of 9,10-dimethyl-1,2-benzanthracene–induced mammary cancer development in rats (6). Chemo-preventive efficacy of SFN was subsequently established inother chemically induced rodent cancer models, includingtobacco carcinogen–induced lung cancer (7), benzo[a]pyrene-induced stomach cancer (8), and azoxymethane-inducedcolonic aberrant crypt foci (9). Previous work from our ownlaboratory has revealed that oral administration of 6 mmolSFN 3 times per week results in significant inhibition of early-stage prostate carcinogenesis in transgenic adenocarcinomaof mouse prostate (TRAMP) mice without any side effects(10). Specifically, the incidence of prostatic intraepithelialneoplasia (PIN) and well-differentiated prostate cancer(WDC) was about 23% to 28% lower in the prostate ofSFN-treated TRAMP mice than in controls (10). However, theincidence of poorly differentiated prostate cancer (PDC) wasnot affected by the SFN regimen used in our study (10). Inanother study, feeding of TRAMPmice with 240 mg of broccolisprout/day resulted in a significant decrease in prostatetumor growth (11). We were the first to show in vivo efficacyof SFN against PC-3 human prostate cancer xenografts inmale athymic mice (12).

Insight into the mechanisms by which SFN likely preventscancer development continues to expand, albeit mostly from

Authors' Affiliations: 1Department of Pharmacology & Chemical Biology,2University of Pittsburgh Cancer Institute, 3Department of Cell Biology andPhysiology, University of Pittsburgh School of Medicine, Pittsburgh; and4Department of Pharmacology, Penn State Milton S. Hershey MedicalCenter, Hershey, Pennsylvania

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

Corresponding Author: Shivendra V. Singh, University of PittsburghCancer Institute, 2.32A Hillman Cancer Center Research Pavilion, 5117Centre Avenue, Pittsburgh, PA 15213. Phone: 412-623-3263; Fax: 412-623-7828; E-mail: [email protected]

doi: 10.1158/0008-5472.CAN-13-0755

�2013 American Association for Cancer Research.

CancerResearch

www.aacrjournals.org 5985

cellular studies. Nevertheless, the mechanisms potentiallycontributing to SFN-mediated chemoprevention includecell-cycle arrest (13), apoptosis induction (14, 15), inhibitionof angiogenesis and histone deacetylase (16, 17), and suppres-sion of oncogenic signaling pathways (e.g., NF-kB, STAT3,and androgen receptor; refs. 18–20). SFN is not a targetedagent as is the case for many other dietary cancer chemo-preventive agents (21).

Previously, we made an exciting observation that exposureof cultured human prostate cancer cells (LNCaP and PC-3) toSFN resulted in induction of autophagy (22), which is anevolutionary conserved physiologic process for bulk degra-dation of macromolecules including organelles and consid-ered a valid cancer therapeutic target (23, 24). We also foundthat autophagy was a protective mechanism against apopto-sis induction by SFN (22). However, the in vivo significance ofthese observations was unclear. The present study addressesthis gap in our knowledge using autophagy inhibitor chlo-roquine (CQ) and the TRAMP mouse model of prostatecancer.

Materials and MethodsReagents and antibodies

SFN was synthesized as described previously (7). The purityof SFN was 98% as determined by high-performance liquidchromatography. Stock solution of SFN was stored at �20�C.SFN was diluted with PBS immediately before use. CQ waspurchased from Sigma-Aldrich. Antibodies against T-antigen,synaptophysin, and E-cadherin were from BD Biosciences;antibodies against microtubule-associated protein 1 lightchain 3 (LC3), androgen receptor (AR), and vimentin werepurchased fromSanta CruzBiotechnology; anti-Ki-67 antibodywas purchased from Dako-Agilent Technologies; and antibo-dies against p62, cleaved caspase-3, Atg5, and phospho-(S2448)-mTOR were purchased from Cell Signaling Technol-ogy. Terminal deoxynucleotidyl transferase–mediated dUTPnick end labeling (TUNEL) staining was conducted usingApopTag Plus Peroxidase In Situ Apoptosis Detection Kit(EMD Millipore). A kit for quantitation of VEGF in tumorlysates was purchased from R&D Systems.

Randomization and treatment of miceUse of mice and their care were in accordance with the

University of Pittsburgh Institutional Animal Care andUse Committee guidelines. Male TRAMP [(C57BL/6xFVB)F1] mice were obtained by crossing TRAMP female in theC57BL/6 background with male FVB mice. Transgene ver-ification was conducted as described by Greenberg andcolleagues (25). After transgene verification, 4-week-oldmale TRAMP mice were placed on AIN-93G diet (HarlanTeklad) for 1 week before the onset of treatments, and themice were maintained on this diet throughout the experi-ment. Initially, as the mice became available from ourbreeding program, a total of 128 mice were placed into oneof the following groups: control (n ¼ 32), CQ alone (n ¼ 32),SFN alone (n ¼ 35), and SFN þ CQ combination (n ¼ 29). Assummarized in Supplementary Table S1, some mice from

each group were removed from the study due to a variety ofreasons, including weight loss, hind limb paralysis, seminalvesicle invasion, and tumors at sites other than prostate.Final number of mice available for evaluations was: control(n ¼ 28), CQ alone (n ¼ 25), SFN alone (n ¼ 28), and SFN þCQ (n ¼ 25). Majority of the evaluable mice in each groupwere treated for 18 weeks but a fraction of mice from control(25%), CQ alone (16%), SFN alone (21%), and SFN þ CQcombination group (8%) were sacrificed after 15 to 17 weeksof treatment due to large tumor burden. Nevertheless, thesemice were included in the analysis. The mice of the controlgroup received 0.1 mL PBS by oral intubation as well asintraperitoneal injection 3 times per week. The CQ alonegroup of mice were treated with 1.2 mg CQ (in 0.1 mL PBS)by intraperitoneal injection and 0.1 mL PBS by oral intuba-tion 3 times per week. The SFN group of mice received 1 mgSFN (in 0.1 mL PBS) by oral intubation and 0.1 mL PBS byintraperitoneal injection 3 times per week. The SFN þ CQgroup of mice received 1 mg SFN (in 0.1 mL PBS) by oralintubation and 1.2 mg CQ (in 0.1 mL PBS) by intraperitonealinjection 3 times per week. Treatments were givenon Monday, Wednesday, and Friday of each week. Bodyweights of the mice were recorded once weekly beginningat 5 weeks of age. The animals were sacrificed by CO2

inhalation followed by cervical dislocation. Blood was col-lected for separation of plasma, which was stored at �20�C.The prostate tissues were harvested, weighed, fixed in10% neutral-buffered formalin, and sectioned at 4 to 5 mmthickness.

Histopathologic evaluationsWholemount hematoxylin and eosin (H&E)-stained sec-

tions of prostate tissues were digitized by scanning and eachsection was blindly scored for microscopic PIN and WDCincidence and burden (affected area). The area of the lesions(in 2 dimensions) was calculated on histologic slides usingon-screen drawing tool of the WebScope viewing softwarefrom Aperio. The mean PIN and WDC area were calculatedfrom sum of the area in an animal and the average of areaover all the animals for each group. Pathologic grading wasconsistent with the criteria defined in our previous studies(10, 26).

ImmunohistochemistryBriefly, prostate sections were quenched with 3% hydrogen

peroxide and blocked with normal serum. The sections werethen probed with the desired primary antibody (anti-synap-tophysin, anti-T-antigen, anti-Ki-67, anti-LC3, anti-AR),washed with Tris-buffered saline and incubated with sec-ondary antibody. Characteristic brown color was developedby incubation with 3,30-diaminobenzidine. The sections werecounterstained with Mayer's hematoxylin (Sigma) and exam-ined under a Leica microscope. The images were analyzedwith the use of ImageScope software (Aperio). Synaptophysinexpression was analyzed using the membrane algorithm,LC3 expression was analyzed using the positive pixel algo-rithm, and nuclear algorithm was used to quantify Ki-67,T-antigen, and AR-positive cells.

Vyas et al.

Cancer Res; 73(19) October 1, 2013 Cancer Research5986

Transmission electron microscopyTransmission electron microscopy using prostate tissue

was conducted for visualization and quantitation of autop-hagic vacuoles. Transmission electron microscopy was con-ducted essentially as described by us previously (26). Briefly,prostate tissues were immersion fixed in 2.5% electronmicroscopy grade glutaraldehyde in PBS overnight at 4�C,washed in PBS, and then post-fixed in 1% aqueous osmiumtetroxide containing 0.1% potassium ferricyanide for 1 hour.Following 3 PBS washes, samples were dehydrated througha graded series of 30% to 100% ethanol. Propylene oxide(100%) was then infiltrated in a 1:1 mixture of propyleneoxide and Poly/Bed 812 epoxy resin (Epon; Polysciences) for1 hour. After several changes of 100% resin over 24 hours,samples were embedded in molds, cured at 37�C overnight,followed by additional hardening at 65�C for 2 days. Ultra-thin (60 nm) sections were collected on 200 mesh coppergrids, stained with 2% uranyl acetate in 50% methanol for 10minutes, and then stained in 1% lead citrate for 7 minutes.Sections were imaged using JEOL 1011 transmission elec-tron microscope at 80 kV fitted with a side-mount digitalcamera.

TUNEL assayProstate sections were deparaffinized, rehydrated, and

then used to visualize apoptotic bodies by TUNEL staining.TUNEL staining was conducted according to the supplier'sinstructions. Four to five randomly selected, nonoverlap-ping, and nonnecrotic fields were imaged to score TUNEL-positive cells.

Western blottingTumor samples from each group were processed for West-

ern blotting as previously described (10). Western blotting wasdone as described previously (26).

Determination of VEGF levelsThe levels of VEGF in tumor lysates from different groups

weremeasured using a commercially available kit according tothe supplier's instructions. The results were normalized toprotein concentration.

Plasma proteomics by 2-dimensional gel electrophoresisand mass spectrometryPlasma proteomics was conducted by Applied Biomics.

Randomly selected plasma samples (without completeknowledge of the histopathologic data) from control mice(n ¼ 3), SFN alone–treated mice (n ¼ 3), and mice treatedwith the SFN þ CQ combination (n ¼ 4) were used forplasma proteomics analyses essentially as described by uspreviously (26). Results are expressed as a ratio of abun-dance of a desired protein in the SFN or SFN þ CQ group tothe control group. Cluster analysis for protein abundancechanges was conducted using a public bioinformatics tool[The Database for Annotation, Visualization and IntegratedDiscovery (DAVID), http://david.abcc.ncifcrf.gov/home.jsp;ref. 27]. The software uses a novel algorithm to measurethe relationships among the annotation terms based on the

degrees of their co-association genes to group the similar,redundant, and heterogeneous annotation contents from thesame or different resources into annotation groups.

Statistical analysisThe association of incidence and treatment was examined

by Fisher exact test. Comparisons of lymph node metastasisor prostate size between the 4 treatment groups wereconducted using Wilcoxon 2-sample test. Area of PIN andWDC was analyzed by one-way ANOVA with Bonferronimultiple comparison adjustment. Statistical significance ofdifferences in mechanistic correlates (immunohistochemi-cal analyses, TUNEL-positive apoptotic bodies, and numberof autophagic vacuoles) was determined using one-wayANOVA followed by Bonferroni adjustment. Statistical anal-yses were conducted using SAS version 9.2 or GraphPadPrism version 4.03. Difference was considered significant atP < 0.05.

ResultsEffects of SFN and/or CQ treatments on PDC

We have shown previously that SFN treatment inducescytoprotective autophagy in cultured human prostate cancercells (PC-3 and LNCaP) regardless of the AR or p53 status (22).The main objective of the present study was to determinethe in vivo significance of these observations. We hypothe-sized that the chemopreventive efficacy of SFN againstprostate cancer might be augmented by pharmacologicinhibition of autophagy using CQ, which disrupts lysosomeacidification (28). As shown in Fig. 1A, the initial and finalbody weights of the mice were not affected by treatmentswith SFN and/or CQ when compared with control. Wetweight of the prostate is an indicator of tumor burden inthe TRAMP model. The mean prostate weight of the micetreated with SFN þ CQ (1.43 g) was lower by about 45%compared with control group (P ¼ 0.02; Fig. 1B). The meanprostate weight of the mice of SFN þ CQ group was alsolower than SFN alone (2.74 g) or CQ alone group (2.47 g), butthe difference did not reach statistical significance (Fig. 1B).Microscopic examinations of the wholemount H&E-stainedsections from the prostate of mice revealed that PDC was thepredominant pathology if the mean prostate weight wasmore than 1 g. We therefore computed percentage of micewith prostate weight of more than 1 g. As shown in Fig. 1C,the percentage of mice with prostate weight more than 1 gwas significantly lower in the SFN þ CQ group (12%) than incontrol (50%), CQ alone (44%), and SFN alone group(54%; Fig. 1C). Collectively, these results indicated inhibitionof PDC upon co-treatment with SFN and CQ, which was notobserved with SFN alone administration.

Effects of SFN and/or CQ treatments on lymph nodemetastasis

Figure 2A depicts metastatic lymph node in a represen-tative mouse each of the control group and the SFN þ CQtreatment group. Lymph node metastasis incidence in theSFN þ CQ–treated mice was lower (12%) than that of the

Chemoprevention of Prostate Cancer by D,L-Sulforaphane

www.aacrjournals.org Cancer Res; 73(19) October 1, 2013 5987

control group (36%), CQ alone group (36%), or the SFN alonegroup (36%; Fig. 2B). The size of the metastatic lymph nodein the mice of SFN þ CQ group was significantly lower (42%lower) than in the control group (Fig. 2C). These resultsindicated that treatment with SFN þ CQ combination wasassociated with inhibition of lymph node metastasis, whichwas not observed by treatment with SFN alone.

Analyses of PIN and WDC incidence/burdenFigure 3A shows pathology associated with low-grade PIN

(LG PIN), high-grade PIN (HG PIN), and WDC in represen-tative TRAMP mice of the control group. Microscopic anal-ysis of PIN and WDC was conducted in prostate of mice with

mean prostate weight less than 1 g because PDC is thepredominant pathology in mice with mean prostate weightmore than 1 g. Neither SFN alone nor SFN þ CQ combina-tion was able to suppress incidence (Fig. 3B) or burden(affected area; Fig. 3C) of LG PIN or HG PIN. On the otherhand, consistent with our previous observations (10), SFNtreatment alone was able to significantly reduce area (79%decrease compared with control, P < 0.01) of WDC comparedwith control (Fig. 3C). Furthermore, the area of the WDC inmice treated with SFN þ CQ combination was about 69%lower than that of control mice (P < 0.01; Fig. 3C). Collec-tively, these results indicated that the effect of SFN þ CQcombination was not superior to SFN alone with respect tothe effect on PIN or WDC incidence and burden.

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Figure 2. SFN and CQ combination treatment decreases lymph nodemetastasis in TRAMP mice. A, microscopic images depicting lymphnode metastasis in a representative mouse each of the control groupand the SFN þ CQ group (magnification, �5; scale bar, 400 mm). B,percentage of mice with lymph node metastasis. Statisticalsignificance was determined by Fisher exact test. C, size of lymphnode metastasis in mice of control group (n ¼ 10), CQ alone group(n ¼ 9), SFN alone group (n ¼ 10), and SFN þ CQ group (n ¼ 3).Results shown are mean � SD. Statistical significance wasdetermined by Wilcoxon 2-sample test.

Vyas et al.


Effects of SFN and/or CQ treatments on neuroendocrinetumorsA small fraction of cells in the TRAMP tumor exhibit

neuroendocrine differentiation (29). Figure 4A depicts synap-tophysin-positive cells (a marker for neuroendocrine cells) ina representative mouse of each group. The number of synap-tophysin-positive cells was not affected by SFN and/or CQtreatments when compared with control (Fig. 4A, bar graph).

SFN treatment caused in vivo autophagyTransmission electron microscopy was conducted for visu-

alization and quantitation of autophagic vacuoles in the pro-state of control mice and those treated with SFN and/or CQ(Fig. 4B). CQ impairs autophagic protein degradation byincreasing intralysosomal pH (28, 30, 31). CQ treatment leadsto accumulation of ineffective autophagosomes, which wasalso observed in the present study (Fig. 4B). It is important topoint out that distinction between effective and ineffectiveautophagosomes is not possible by electron microscopy. Nev-ertheless, the number of autophagic vacuoles was significantly

higher in the prostate of SFN-treated mice compared withcontrol (Fig. 4B). Autophagy induction by SFN administrationin vivo was confirmed by immunohistochemical analysis ofLC3, which is a critical protein in autophagic machinery (32).Expression of LC3 was relatively higher in the prostate of micetreated with SFN alone and SFN þ CQ compared with controlbut the difference did not reach statistical significance (Fig.4C). Nevertheless, these observations provided in vivo evidencefor SFN-induced autophagy.

Analysis of Ki-67 and AR expression and TUNEL-positiveapoptotic bodies

We raised the question of whether SFN-mediated preven-tion of prostate cancer in TRAMP model was partly due tosuppression of the transgene. Expression of T-antigen wasnot affected by SFN and/or CQ treatments (Fig. 5A). Whilethe expression of Ki-67 (a marker of cell proliferation)was significantly lower in the tumors of SFN alone andSFN þ CQ group than in control or CQ alone group (Fig.5B), the AR expression did not differ significantly between

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Figure 3. Effects of SFN and/orCQ treatments on incidence andburden (affected area) of LG PIN,HGPIN, andWDC in TRAMPmice.A, microscopic images depictingpathology associated with LG PIN,HG PIN, and WDC (magnification,�200; scale bar, 50 mm). B,incidence of LG PIN, HG PIN, andWDC in the prostate of controlmice and those treated with SFNand/or CQ. Statistical significancewas determined by Fisher exacttest. C, area of LG PIN, HG PIN,and WDC in the prostate of micefrom the control group (n¼ 13), CQalone group (n ¼ 12), SFN alonegroup (n¼9), andSFNþCQgroup(n ¼ 20). Results shown aremean � SD. Statisticalsignificance was determined byone-way ANOVA followed byBonferroni multiple comparisontest.



groups (Fig. 5C). The TUNEL-positive apoptotic bodies wereless in the prostate of control mice or those treated with CQ(Fig. 5D). The number of TUNEL-positive apoptotic bodieswas increased upon treatment with SFN compared with CQ.The SFN þ CQ combination was even more effective inincreasing the number of TUNEL-positive cells but thedifference was insignificant due to large variability.

We proceeded to determine the expression of p62, which isanother marker of autophagy, and caspase-3 cleavage, which isan indicator of apoptosis, using tumor supernatants from 3 to 4mice of each group. Two of 4 samples showed decreasedintensity for p62 expression in the SFN þ CQ group whencompared with control (Supplementary Fig. S1). Cleavage ofcaspase-3 was very low in tumors of control and CQ alonegroups as expected but was increased in SFN alone and SFNþ

CQ groups attesting to apoptosis induction by these treat-ments (Supplementary Fig. S1).

Analysis of E-cadherin, vimentin, Atg5, phospho-mTOR,and VEGF proteins

Epithelial–mesenchymal transition (EMT), biochemicallycharacterized by loss of adherens junction protein E-cadherinconcomitant with induction of vimentin, is implicated as asignificant contributor to cancer progression as well as met-astatic spread (33, 34). The expression of E-cadherin proteinwas either undetectable or very low in the tumors of control,CQ alone, and SFN alone treatment groups. On the other hand,the expression of E-cadherinwas clearly visible in some tumorsof mice from the SFNþ CQ treatment group (Fig. 6A). Becauseof large variability in E-cadherin expression and less than

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Figure 4. Effects of SFN and/orCQ treatments on synaptophysinexpression (magnification, �200;scale bar, 50 mm; A), autophagicvacuoles quantified fromtransmission electronmicrographs (magnification,�25,000; scale bar, 500 nm; B),and LC3 expression(magnification, �200; scale bar,50mm;C) in the prostate of TRAMPmice. Results shown are mean �SD (n ¼ 7 for each group in Aand C). For quantitation ofautophagic vacuoles, prostatetissues from 2 mice of each groupwere used for transmissionelectron microscopy, and entirefields from 3 to 6 sections for eachsample were examined forpresence of autophagic vacuoles.Results shown in B are mean� SD(n ¼ 6–9). Statistical significancewas determined by one-wayANOVA followed by Bonferronimultiple comparison test.

Vyas et al.


sufficient power because of a profound effect of the SFNþ CQcombination on prostate cancer development, statistical anal-ysis was not possible (Fig. 6B). Nevertheless consistent with theE-cadherin protein expression data, the level of vimentinprotein was lower in the tumors of SFN þ CQ group incomparison with other groups (Fig. 6A). Thus, potential inhi-bition of EMT in the tumor by the SFN þ CQ combinationtreatment likely contributes to its anti-metastatic effect. Like-wise, there was a trend of an increase in protein levels ofautophagy regulator Atg5 in the tumors of SFN þ CQ–treatedmice compared with control or SFN treatment groups (Fig. 6Aand B). mTOR is another protein implicated in regulation ofautophagy (35). Protein level of phospho-mTOR (active form)was relatively lower in the tumors of SFN þ CQ–treated micethan in the tumors of control mice. Finally, SFN þ CQ com-

bination also resulted in a modest decrease in protein levels ofangiogenic cytokine VEGF (36) in the tumor in comparisonwith those of control and CQ groups of mice (Fig. 6C). Theseobservations provide mechanistic insights into the chemo-preventive effect of the SFN þ CQ combination.

Changes in plasma protein abundance upon treatmentwith SFN and SFN þ CQ

The cutoff criteria for spot selection and protein identifica-tion were at least 1.3-fold difference (increase or decrease) inabundance of different proteins in the plasma of mice fromSFNþ CQ group compared with control and P� 0.10. Some ofthe spots meeting these criteria were abundant proteins (e.g.,hemoglobin, macroglobulin, etc.) even though depletion ofabundant proteins was conducted before the 2-dimensional

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Figure 5. Effects of SFN and/orCQ treatments on T-antigen andAR expression and markers ofproliferation and apoptosis.A, T-antigen expression(magnification, �200; scalebar, 50 mm). B, Ki-67 expression(magnification, �200; scalebar, 50 mm). C, AR expression(magnification, �200; scale bar,50 mm). D, TUNEL-positiveapoptotic bodies in the prostate ofTRAMPmice (magnification,�200;scale bar, 50 mm). Results shownare mean � SD (n ¼ 7 for eachgroup). Statistical significance wasdetermined by one-way ANOVAfollowed by Bonferroni multiplecomparison test.



gel electrophoresis. The abundant proteins are not includedin Table 1, which summarizes plasma protein abundancechanges in response to treatment with SFN alone or SFN þCQ combination in comparison with control. Cluster analysisof the proteomics results indicated significant enrichment ofproteins associated with proteasome, protease inhibitor fam-ily, and protein–lipid complex (results not shown).

DiscussionOur initial observations of autophagy induction by SFN

treatment in cultured prostate cancer cells (22, 37) have sincebeen confirmed by other investigators in different types ofcancer cells (38–41). Majority of these studies support ourconclusions that autophagy is a cytoprotective mechanism(38–40). For example, pharmacologic inhibition of autophagyusing 3-methyl adenine or bafilomycin A1 augments SFN-

induced apoptotic cell death in cultured human colon andbreast cancer cells (38, 40). However, the in vivo significance ofthese findings was unclear. The present study is the firstpublished report to show that (i) SFN administration causesin vivo autophagy as evidenced by transmission electronmicroscopy and immunohistochemical analyses of LC3 ex-pression and (ii) inhibition of autophagy by an agent (CQ)already under clinical investigation as a chemotherapy sensi-tizer (30) increases efficacy of SFN for prevention of prostatecancer reflected by significant inhibition of PDC by the SFNþCQ combination, which is not observed with SFN treatmentalone. These observations merit investigation of the SFNþ CQcombination for prevention of prostate cancer in a clinicalsetting.

We have shown previously that the incidence of meta-static nodules in the lung, but not the lymph node metas-tasis, is decreased upon treatment with SFN alone (10). In

E-cadherin

Vimentin

Atg5

P-mTOR

Actin

Actin

Actin

Actin

Mouse# 1 2 3 1 2 3 1 2 3 1 2 3


Mouse# 1 2 3 1 2 3 1 2 3 1 2 3 4


Mouse# 1 2 3 1 2 3 1 2 3 1 2 3 4


Mouse# 1 2 3 1 2 3 1 2 3 1 2 3


BA

Cont. CQ SFN SFN+CQ0

50

100

150

n = 6 n = 6 n = 6

n = 5

E-c

adheri

n (

Arb

itra

ry u

nits)

Cont. CQ SFN SFN+CQ0.0

0.5

1.0

1.5

2.0

n = 5n = 6 n = 6

n = 6

Vim

entin (

Arb

itra

ry u

nits)


3

6

9

12

15

18

n = 6

n = 6

n = 6

n = 7

Atg

5 (

Arb

itra

ry u

nits)

Cont. CQ SFN SFN+CQ0.0

0.5

1.0

1.5

2.0

n = 6n = 6

n = 6n = 5

p-m

TO

R (

Arb

itra

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C


100

200

300

400 n = 3

n = 3

n = 3n = 4

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GF

Concentr

atio

n(p

g/m

g p

rote

in)

Figure 6. Effects of SFN and/orCQ treatments on E-cadherin,vimentin, Atg5, phospho-mTOR,and VEGF protein levels. A,Western blotting for E-cadherin,vimentin, Atg5, and phospho-mTOR using tumor lysates frommice of the control group (n ¼ 5–6)and those treated with SFN alone(n¼6), CQalone (n¼6), andSFNþCQ combination (n ¼ 5–7). Resultsshown are mean � SD. B,quantitation of theWestern blottingdata. C, levels of VEGF in tumorlysates from mice of the controlgroup and those treated withSFN and/or CQ.

Vyas et al.


the present study, the overall incidence of lung metastasis incontrol mice was surprisingly lower (<10%) than observedby us previously (>75%) in TRAMP model (10, 42). On theother hand, the overall incidence of lymph node metastasisobserved in control mice in the present study was compa-rable to those reported previously (10, 42). The present studyreveals that the incidence as well as size of lymph nodemetastasis is significantly reduced upon treatment with SFNþ CQ combination when compared with control group. Themechanism by which SFN þ CQ combination suppressesmetastasis likely involves inhibition of EMT and suppressionof proangiogenic cytokine VEGF. We have shown previouslythat SFN treatment alone is unable to restore E-cadherinexpression (10). Nevertheless, further studies are needed tovalidate these conclusions with larger sample size, whichwas not possible in the present study.The SFNþ CQ combination regimen is not superior to SFN

alone with respect to inhibition of PIN or WDC. Consistentwith our previous observations (10), SFN alone is able tomodestly and significantly inhibit WDC incidence and burden,respectively. However, effect on WDC incidence is abolishedwhen SFN is combined with CQ. It is possible that SFN þ CQcombination is more effective in inducing apoptosis in fullytransformed cells (PDC) than in preneoplastic cells (e.g., PIN).This speculation is consistent with our earlier observationsthat normal human prostate epithelial cells are resistant to

SFN-induced apoptosis (15). Relatively greater abundance ofTUNEL-positive apoptotic cells and increased cleavage ofcaspase-3 in the tumors from mice treated with the SFN þCQ combination compared with SFN alone treatment group(present study) provides added support to this contention.

We have shown previously that SFN treatment causestranscriptional repression of AR in human prostate cancercells, leading to downregulation of AR-regulated gene productprostate-specific antigen (20). The present study reveals thatlevel of AR protein in vivo is not decreased upon treatmentwith SFN alone or SFN þ CQ combination. It is possible thata more intense dosing regimen of SFN administration (i.e.,daily administration or higher dose) is required to observesuppression of AR protein expression in vivo. At the same time,the possibility that SFN-mediated suppression of AR signalingis an in vitro phenomenon cannot be discarded.

Successful implementation of a cancer chemopreventivestrategy is contingent upon systematic investigations start-ing with cellular studies to identify promising agents andto define the mechanism(s) underlying their cancer protec-tive effect to animal-based studies focusing not only on invivo bioavailability, safety, and efficacy assessments but alsoon identification of biomarker(s) predictive of tissue expo-sure, and possibly response, before translation in humanswith a pilot biomarker modulation trial followed by largerstudies with cancer incidence as the primary end point.

Table 1. Plasma proteins altered by SFN or SFN þ CQ administration in TRAMP mice

SFN/control SFN þ CQ/control

Protein nameMasterno. P Ratio P Ratio

Serine (or cysteine) peptidase inhibitor, clade A, member3K [Mus musculus]

83 0.600 1.1 0.062 �1.3

Hemopexin precursor [Mus musculus] 195 0.590 �1.1 0.035 �1.5Transferrin [Mus musculus] 201 0.620 �1.1 0.021 �1.4Hemopexin, partial [Mus musculus] 212 0.500 1.2 0.073 �1.4Transferrin 229 0.950 1.0 0.093 �1.3Kininogen-1 isoform 1 precursor [Mus musculus] 277 0.850 1.0 0.031 �1.3Alpha-1-acid glycoprotein 1 precursor [Mus musculus] 529 0.640 1.7 0.049 �2.6Apolipoprotein A-IV precursor [Mus musculus] 561 0.760 1.0 0.031 �1.3Serpina1c protein [Mus musculus] 766 0.960 1.0 0.077 1.6Sulfated glycoprotein-2 isoform 2 [Mus musculus] 860 0.440 1.2 0.051 1.7Proteasome subunit alpha type-1 [Mus musculus] 929 0.780 1.0 0.011 �1.7Transthyretin precursor [Mus musculus] 989 0.500 �1.2 0.004 �2.0Unnamed protein product [Mus musculus] 1074 0.760 1.1 0.047 �1.8Proteasome subunit alpha type-4 [Mus musculus] 1033 0.740 1.1 0.090 1.9Spermatogenesis-associated protein 22 OS ¼ Mus musculusGN ¼ Spata22 PE ¼ 2 SV ¼ 1

1070 0.350 �1.3 0.006 �2.4

Proteasome subunit alpha type-6 [Mus musculus] 1096 0.250 �1.4 0.008 �2.0Transthyretin precursor [Mus musculus] 1592 0.980 1.0 0.090 �1.8Transthyretin precursor [Mus musculus] 1667 0.850 1.0 0.083 �1.6Transthyretin precursor [Mus musculus] 1687 0.410 1.1 0.016 1.3Chain A, crystal structure Of mouse transthyretin 2134 0.100 �1.1 0.100 1.8Apolipoprotein A-II precursor [Mus musculus] 2383 0.520 1.2 0.079 1.3Apolipoprotein A-II precursor [Mus musculus] 2435 0.270 1.2 0.003 1.4



Identification of suitable biomarker(s) is particularly essen-tial for the clinical development of chemopreventive agentsas cancer incidence is too rigorous of an end point formalignancies with long latency such as prostate cancer.Plasma proteomics conducted in the present study providesnovel leads that can be followed up to identify biomarkersassociated with SFN þ CQ exposure/response. Abundancechange of some of these proteins seems unique to the SFN þCQ combination. For example, abundance of alpha 1 acidglycoprotein, which is an acute inflammatory biomarker thatincreases in various conditions including malignancy (43), isdecreased after SFN þ CQ treatment (2.6-fold decreasecompared with control) but its level increases in the plasmaof SFN alone treated mice. We observed decreased abun-dance of kininogen-1 isoform 1 precursor and transthyretinprecursor in the plasma of SFN þ CQ–treated mice com-pared with control. Abundance of these proteins is notaltered by SFN administration alone (present study). Inter-estingly, transthyretin and high molecular weight kininogenwere found to be significantly enhanced in the sera of pati-ents with prostate cancer compared with those of benignprostatic hyperplasia (44).

We found increased abundance of sulfated glycoprotein-2isoform 2 in the plasma of SFN þ CQ–treated mice comparedwith control. This protein is also known as clusterin andexpressed as 3 forms with different subcellular localization(45). Expression of clusterin was shown to be downregulatedduring prostate cancer progression in the TRAMP model (46).Mice with homozygous or heterozygous deletion of clusterinexhibited PIN or differentiated carcinoma (47). Crossing ofclusterin knockout mice with TRAMP resulted in a strongenhancement of metastatic spread, suggesting a tumor sup-pressor role for clusterin (47). At the same time, some studieshave suggested oncogenic function for clusterin (48). Never-theless, SFN þ CQ combination restores plasma level ofisoform 2 of clusterin, and this effect is not as pronouncedupon treatment with SFN alone.

Men with prostate cancer suffer significant impairments inquality of life not only from the disease itself but also as aconsequence of the treatments. Because the commonly asso-ciated risk factors for prostate cancer, including age, race, andgenetic predisposition are not easily modifiable, novelapproaches for prevention of this disease are desirable. More-over, a clinically viable preventive intervention against prostatecancer is still lacking (49, 50). For example, increased incidenceof high-grade tumors in the treatment arm in chemopreven-tion trials with 5-a-reductase inhibitors hindered their broadacceptance as a preventive strategy (49, 50). The results ofthe present study provide preclinical in vivo evidence forprostate cancer preventive efficacy of a novel and clinicallyviable combination regimen to warrant investigations in aclinical setting.

Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.

Authors' ContributionsConception and design: D. Desai, S.V. SinghDevelopment of methodology: E.R. Hahm, D. Desai, S. AminAcquisition of data (provided animals, acquired andmanaged patients, pro-vided facilities, etc.): A.R. Vyas, E.R. Hahm, J.A. Arlotti, S. Watkins, D. Beer-StolzAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): A.R. Vyas, E.R. Hahm, J.A. Arlotti, D. Beer-Stolz,S. Amin, S.V. SinghWriting, review and/or revision of the manuscript: A.R. Vyas, E.R. Hahm,D. Desai, S. Amin, S.V. SinghStudy supervision: S.V. Singh

Grant SupportThis work was supported by the grant RO1 CA115498-07 awarded by the

National Cancer Institute. This research used the Animal Facility and Tissue andResearch Pathology Facility supported in part by a grant from the NationalCancer Institute at the NIH (P30 CA047904).

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

Received March 15, 2013; revised June 27, 2013; accepted July 14, 2013;published OnlineFirst August 6, 2013.

References1. Ambrosone CB, McCann SE, Freudenheim JL, Marshall JR, Zhang

Y, Shields PG. Breast cancer risk in premenopausal women isinversely associated with consumption of broccoli, a source ofisothiocyanates, but is not modified by GST genotype. J Nutr 2004;134:1134–8.

2. Kolonel LN, Hankin JH,Whittemore AS,WuAH, Gallagher RP,WilkensLR, et al. Vegetables, fruits, legumes and prostate cancer: a multi-ethnic case-control study. Cancer Epidemiol Biomarkers Prev 2000;9:795–804.

3. Siegel R, Naishadham D, Jemal A. Cancer statistics, 2012. CA CancerJ Clin 2012;62:10–29.

4. Fahey JW, Zalcmann AT, Talalay P. The chemical diversity and dis-tribution of glucosinolates and isothiocyanates among plants. Phyto-chemistry 2001;56:5–51.

5. Singh SV, Singh K. Cancer chemoprevention with dietary isothiocya-nates mature for clinical translational research. Carcinogenesis 2012;33:1833–42.

6. Zhang Y, Kensler TW, Cho CG, Posner GH, Talalay P. Anticarci-nogenic activities of sulforaphane and structurally related syntheticnorbornyl isothiocyanates. Proc Natl Acad Sci U S A 1994;91:3147–50.

7. Conaway CC, Wang CX, Pittman B, Yang YM, Schwartz JE, Tian D,et al. Phenethyl isothiocyanate and sulforaphane and their N-acet-ylcysteine conjugates inhibit malignant progression of lung adenomasinduced by tobacco carcinogens in A/J mice. Cancer Res 2005;65:8548–57.

8. Fahey JW, Haristoy X, Dolan PM, Kensler TW, Scholtus I, StephensonKK, et al. Sulforaphane inhibits extracellular, intracellular, and antibi-otic-resistant strains of Helicobacter pylori and prevents ben-zo[a]pyrene-induced stomach tumors. Proc Natl Acad Sci U S A2002;99:7610–5.

9. Chung FL, Conaway CC, Rao CV, Reddy BS. Chemoprevention ofcolonic aberrant crypt foci in Fischer rats by sulforaphane and phe-nethyl isothiocyanate. Carcinogenesis 2000;21:2287–91.

10. Singh SV, Warin R, Xiao D, Powolny AA, Stan SD, Arlotti JA, et al.Sulforaphane inhibits prostate carcinogenesis and pulmonary metas-tasis in TRAMP mice in association with increased cytotoxicity ofnatural killer cells. Cancer Res 2009;69:2117–25.

11. Keum YS, Khor TO, Lin W, Shen G, Kwon KH, Barve A, et al.Pharmacokinetics and pharmacodynamics of broccoli sprouts onthe suppression of prostate cancer in transgenic adenocarcinoma ofmouse prostate (TRAMP) mice: implication of induction of Nrf2, HO-1

Vyas et al.


and apoptosis and the suppression of Akt-dependent kinase pathway.Pharm Res 2009;26:2324–31.

12. Singh AV, Xiao D, Lew KL, Dhir R, Singh SV. Sulforaphane inducescaspase-mediated apoptosis in cultured PC-3 human prostate cancercells and retards growth of PC-3 xenografts in vivo. Carcinogenesis2004;25:83–90.

13. Singh SV, Herman-Antosiewicz A, Singh AV, Lew KL, Srivastava SK,Kamath R, et al. Sulforaphane-induced G2/M phase cell cycle arrestinvolves checkpoint kinase 2-mediated phosphorylation of cell divi-sion cycle 25C. J Biol Chem 2004;279:25813–22.

14. Singh SV, Srivastava SK, Choi S, Lew KL, Antosiewicz J, Xiao D,et al. Sulforaphane-induced cell death in human prostate cancercells is initiated by reactive oxygen species. J Biol Chem 2005;280:19911–24.

15. Choi S, Singh SV. Bax and Bak are required for apoptosis induction bysulforaphane, a cruciferous vegetable-derived cancer chemopreven-tive agent. Cancer Res 2005;65:2035–43.

16. Bertl E, Bartsch H, Gerh€auser C. Inhibition of angiogenesis andendothelial cell functions are novel sulforaphane-mediated mechan-isms in chemoprevention. Mol Cancer Ther 2006;5:575–85.

17. MyzakMC, Karplus PA, Chung FL, DashwoodRH. A novelmechanismof chemoprotection by sulforaphane: inhibition of histone deacetylase.Cancer Res 2004;64:5767–74.

18. XuC, ShenG,ChenC, G�elinas C, Kong AN. Suppression of NF-kBandNF-kB-regulated gene expression by sulforaphane andPEITC throughIkBa, IKK pathway in human prostate cancer PC-3 cells. Oncogene2005;24:4486–95.

19. Hahm ER, Singh SV. Sulforaphane inhibits constitutive and inter-leukin-6-induced activation of signal transducer and activator oftranscription 3 in prostate cancer cells. Cancer Prev Res 2010;3:484–94.

20. Kim SH, Singh SV. D,L-Sulforaphane causes transcriptional repres-sion of androgen receptor in human prostate cancer cells. Mol CancerTher 2009;8:1946–54.

21. Surh YJ. Cancer chemoprevention with dietary phytochemicals. NatRev Cancer 2003;3:768–80.

22. Herman-Antosiewicz A, Johnson DE, Singh SV. Sulforaphane causesautophagy to inhibit release of cytochrome c and apoptosis in humanprostate cancer cells. Cancer Res 2006;66:5828–35.

23. Dikic I, Johansen T, Kirkin V. Selective autophagy in cancer develop-ment and therapy. Cancer Res 2010;70:3431–4.

24. Kondo Y, Kanzawa T, Sawaya R, Kondo S. The role of autophagy incancer development and response to therapy. Nat Rev Cancer 2005;5:726–34.

25. Greenberg NM, DeMayo F, FinegoldMJ,Medina D, TilleyWD, AspinallJO, et al. Prostate cancer in a transgenic mouse. Proc Natl Acad SciU S A 1995;92:3439–43.

26. Powolny AA, Bommareddy A, Hahm ER, Normolle DP, Beumer JH,Nelson JB, et al. Chemopreventative potential of the cruciferousvegetable constituent phenethyl isothiocyanate in a mouse model ofprostate cancer. J Natl Cancer Inst 2011;103:571–84.

27. Huang DW, Sherman BT, Lempicki RA. Systematic and integrativeanalysis of large gene lists using DAVID bioinformatics resources. NatProtoc 2009;4:44–57.

28. Michihara A, Toda K, Kubo T, Fujiwara Y, Akasaki K, Tsuji H. Disruptiveeffect of chloroquine on lysosomes in cultured rat hepatocytes. BiolPharm Bull 2005;28:947–51.

29. Chiaverotti T, Couto SS, Donjacour A, Mao JH, Nagase H, Cardiff RD,et al. Dissociation of epithelial and neuroendocrine carcinomalineages in the transgenic adenocarcinoma of mouse prostate modelof prostate cancer. Am J Pathol 2008;172:236–46.

30. Hu YL, Jahangiri A, DeLay M, Aghi MK. Tumor cell autophagy as anadaptive response mediating resistance to treatments such as anti-angiogenic therapy. Cancer Res 2012;72:4294–9.

31. Amaravadi RK, Yu D, Lum JJ, Bui T, Christophorou MA, Evan GI, et al.Autophagy inhibition enhances therapy-induced apoptosis in a Myc-induced model of lymphoma. J Clin Invest 2007;117:326–36.

32. Kabeya Y, Mizushima N, Ueno T, Yamamoto A, Kirisako T, Noda T,et al. LC3, a mammalian homologue of yeast Apg8p, is localized inautophagosome membranes after processing. EMBO J 2000;19:5720–8.

33. Thiery JP. Epithelial-mesenchymal transitions in tumour progression.Nat Rev Cancer 2002;2:442–54.

34. Hugo H, Ackland ML, Blick T, Lawrence MG, Clements JA, WilliamsED, et al. Epithelial-mesenchymal and mesenchymal-epithelial transi-tions in carcinoma progression. J Cell Physiol 2007;213:374–83.

35. Pyo JO, Nah J, Jung YK. Molecules and their functions in autophagy.Exp Mol Med 2012;44:73–80.

36. McMahonG. VEGF receptor signaling in tumor angiogenesis. Oncolo-gist 2000;5 Suppl 1:3–10.

37. XiaoD,PowolnyAA,Antosiewicz J,HahmER,BommareddyA, ZengY,et al. Cellular responses to cancer chemopreventive agent D,L-sulfo-raphane in human prostate cancer cells are initiated by mitochondrialreactive oxygen species. Pharm Res 2009;26:1729–38.

38. Nishikawa T, Tsuno NH, Okaji Y, Shuno Y, Sasaki K, Hongo K, et al.Inhibitionof autophagypotentiates sulforaphane-inducedapoptosis inhuman colon cancer cells. Ann Surg Oncol 2010;17:592–602.

39. JeongHS,Choi HY, Lee ER, Kim JH, JeonK, LeeHJ, et al. Involvementof caspase-9 in autophagy-mediated cell survival pathway. BiochimBiophys Acta 2011;1813:80–90.

40. Kanematsu S, Uehara N, Miki H, Yoshizawa K, Kawanaka A, Yuri T,et al. Autophagy inhibition enhances sulforaphane-induced apoptosisin human breast cancer cells. Anticancer Res 2010;30:3381–90.

41. Naumann P, Fortunato F, Zentgraf H, B€uchler MW, Herr I, Werner J.Autophagy and cell death signaling following dietary sulforaphane actindependently of each other and require oxidative stress in pancreaticcancer. Int J Oncol 2011;39:101–9.

42. SinghSV, Powolny AA, Stan SD, XiaoD, Arlotti JA,Warin R, et al. Garlicconstituent diallyl trisulfide prevents development of poorly differen-tiated prostate cancer and pulmonary metastasis multiplicity inTRAMP mice. Cancer Res 2008;68:9503–11.

43. Kanoh Y, Ohtani H, Egawa S, Baba S, Akahoshi T. Levels of acuteinflammatory biomarkers in advanced prostate cancer patients witha2-macroglobulin deficiency. Int J Oncol 2011;39:1553–8.

44. Jayapalan JJ, Ng KL, Razack AH, Hashim OH. Identification of poten-tial complementary serum biomarkers to differentiate prostate cancerfrom benign prostatic hyperplasia using gel- and lectin-based prote-omics analyses. Electrophoresis 2012;33:1855–62.

45. Rizzi F, Bettuzzi S. The clusterin paradigm in prostate and breastcarcinogenesis. Endocr Relat Cancer 2010;17:R1–17.

46. Caporali A,Davalli P, Astancolle S, D'ArcaD, BrausiM,Bettuzzi S, et al.The chemopreventive action of catechins in the TRAMPmouse modelof prostate carcinogenesis is accompanied by clusterin over-expres-sion. Carcinogenesis 2004;25:2217–24.

47. Bettuzzi S, Davalli P, Davoli S, Chayka O, Rizzi F, Belloni L, et al.Genetic inactivation of ApoJ/clusterin: effects on prostate tumouri-genesis and metastatic spread. Oncogene 2009;28:4344–52.

48. Miyake H, Muramaki M, Kurahashi T, Yamanaka K, Hara I, Gleave M,et al. Expression of clusterin in prostate cancer correlateswithGleasonscore but not with prognosis in patients undergoing radical prosta-tectomy without neoadjuvant hormonal therapy. Urology 2006;68:609–14.

49. Thompson IM, Goodman PJ, Tangen CM, Lucia MS, Miller GJ, FordLG, et al. The influence of finasteride on the development of prostatecancer. N Engl J Med 2003;349:215–24.

50. Andriole GL, Bostwick DG, Brawley OW, Gomella LG, Marberger M,Montorsi F, et al. Effect of dutasteride on the risk of prostate cancer.N Engl J Med 2010;362:1192–202.



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