The Prostate 68:345 ^353 (2008)

The ImpactofDiet andMicronutrient Supplementsonthe ExpressionofNeuroendocrineMarkers in

Murine LadyTransgenic Prostate

Jodie Palmer,1 Vasundara Venkateswaran,2 Neil E. Fleshner,3

Laurence H. Klotz,2 and Michael E. Cox1,4*1The Prostate Centreat VancouverGeneralHospital,Vancouver,British Columbia,Canada

2DivisionofUrology, SunnybrookHealth Sciences Centre,Toronto,Ontario,Canada3DivisionofUrology, PrincessMargaretHospital,Toronto,Ontario,Canada

4DepartmentofUrological Sciences,Universityof British Columbia,Vancouver,British Columbia,Canada

BACKGROUND. Neuroendocrine (NE) differentiation (NED) in prostate cancer (PCa) isassociatedwithmorbidity and death; however, the underlying cause(s) promotingNED in PCahave yet to be determined. In this study, we examined the effect of both diet andmicronutrientsupplementation on the expression of NEmarkers using the Lady (12T-10) transgenic model ofPCa. Lady (12T-10) transgenic animals develop advanced adenocarcinoma with NE character-istics that exhibits metastases in approximately 80% of cases. In this model a high fat diet hasbeen shown to increase the severity of disease, while the use of micronutrients can inhibit thisprogression.METHODS. In this study we used immunohistochemical analysis to determine expression oftheNEmarkers: chromograninA (CgA), neuron-specific enolase (NSE), bombesin, parathyroidhormone-related peptide (PTHrP), neurotensin and serotonin in prostates of PCa-bearing Lady(12T-10) mice.RESULTS. High fat diet was correlated with significantly elevated expression of CgA andserotonin in prostate tissue of Lady (12T-10) mice. Addition of micronutrients to the control andhigh fat diet reproducibly elevated PTHrP and bombesin expression and suppressed NSEexpression, while prostate tissue from the control diet supplemented with micronutrientsexhibited significantly lower numbers of calcitonin- and neurotensin-positive cells.CONCLUSIONS. These results highlight the importance of dietary control in management ofdisease and identify differential changes inNEmarker expression,whichmaybediagnosticallyviable in monitoring the impact of therapies on disease status. Prostate 68: 345–353, 2008.# 2008 Wiley-Liss, Inc.

KEY WORDS: prostate cancer; neuroendocrine; dietary; transgenic; CgA

INTRODUCTION

Prostate cancer (PCa) remains the most frequentlydiagnosed malignancy and the third leading cause ofcancer related death in men in western countries [1].Genetic profile, environment and diet have all beenimplicated in PCa incidence and disease progression[1–5]. Evidence of the negative effect of a high fat dieton the progression of prostate disease and the use ofantioxidants to manage prostate disease is mounting[4–9]. The heterogeneity of the disease has led tothe development of mouse models of PCa, includingTRAMP and Lady transgenics, as genomic instabilitymodels to better understand and manipulate disease

Abbreviations: PCa, prostate cancer; CgA, Chromogranin A; NSE,neuron specific enolase; PTHrP, parathyroid hormone related peptide.

Grant sponsor: JP, BC Foundation for Prostate Disease; Grantsponsor: VV, CIHR; Grant sponsor: NEF & LHK, Canadian ProstateCancer Bio-Research Network; Grant sponsor: MEC, NationalCancer Institute of Canada; Grant sponsor: Michael Smith Founda-tion for Health Research.

Jodie Palmer’s present address is Ludwig Institute for CancerResearch, Austin Health, Heidelberg, VIC, Australia.

*Correspondence to: Michael E. Cox, The Prostate Centre atVancouver General Hospital, 2660 Oak St, Vancouver, BC, CanadaV6H 3Z6. E-mail: mcox@interchange.ubc.caReceived 23 July 2007; Accepted 27 September 2007DOI 10.1002/pros.20692Published online 10 January 2008 in Wiley InterScience(www.interscience.wiley.com).

� 2008 Wiley-Liss, Inc.

initiation, progression and morbidity [10,11]. Theseand other T-antigen-driven murine PCa modelshave been characterized to express various cytologicmarkers associated with Neuroendocrine differentia-tion (NED) [12], though the significance of thesephenotypic features is unknown.

NE-like cells are found in the normal prostate aswellas in benign prostate lesions where they are thought toparticipate in organogenesis and tissue homeostasis[13–15]. In neoplasias their prevalence correlates withtumor size and progression to hormone-resistant states[16–20]. Several clinical studies have concluded thatprostatic NE-like cells and their associated markersserve as prognostic indicators of poor patient survival[21–26] following the characterization of an arrayof markers including: S-100, chromogranin A (CgA),neuron-specific enolase (NSE), and neurosecretoryfactors such as bombesin, parathyroid hormone-related peptide (PTHrP), serotonin and neurotensin[12]. NE markers of greatest interest are those asso-ciated with AI progression, which may allow earlydetection of high-risk patients.NSEandCgAhave beenrecognized clinically by both serum and histocyto-chemical testing of biopsies to be of interest inrecognition of early and advanced PCa, respectively,as well as non-PSA reactive disease [26–29]. While aspecific role of these in the prostate has not beenidentified, classically the enolases play an importantrole in the end stages of glycolysis while the graninsregulate secretion [30], functions important in cell–cellsignaling and paracrine growth factor release in thedeveloping tumor. Less well characterized factors:bombesin, serotonin, and neurotensin, have beendemonstrated as both mitogens and inducers ofmigration in vitro [31,32], while PTHrP and calcitoninmay be important in the development of metastasesdue to their ability to regulate osteoclastogenesis andosteblastogenesis [33–35]. Several of these factors havebeen attributed with growth and survival of PCa cellline models [36–38]. For example, LNCaP xenograftsseeded with NE differentiated cells progress morerapidly to androgen independence [39], supporting thenotion that NED in PCa promotes disease progression.

We have utilized Lady (12T-10) transgenic [11] malemice to investigate the effect of diet and micronutrientuse on the NED of prostate cells. Mice on a control dietexhibited extensive invasive and metastatic adenocar-cinoma at 32 weeks of age (15/19mice; 73% incidence).High-fat diet potentiated tumor development (19/19mice; 100% incidence) while the addition of the micro-nutrients selenium, vitamin E, and lycopene in the dietsignificantly inhibited tumor development in morethan 80% of animals (pathology observed in 10.53% ofanimals (2/19) with a control diet, 15.79% of animals(3/9) on a high fat diet supplemented with micro-

nutrients) [40]. We examined the expression of theneuroendocrine (NE) markers CgA, NSE, Bombesin,Serotonin, PTHrP, neurotensin, and calcitonin inprostate tissue from this study. Of animals examined,all control and high fat diet animals had invasivecarcinoma, while micronutrient treated animals hadno evidence of invasive carcinoma, and <20% of theseanimals had evidence of hyperplasia. These animalswere examined in an attempt to clarify the role diet andmicronutrients play in disease progression of Ladytransgenics.

MATERIALSANDMETHODS

TransgenicMice

The Lady (12T-10) probasin promoter-driven LargeT-antigen transgenic mouse line developed in a CD1background were obtained from Dr. Robert J. Matusik(Vanderbilt Prostate Center, Vanderbilt UniversityMedical Center, Nashville, TN) [11,41]. Breeding pairswere fed control diet of standard pellet mouse feed.After weaning at 3–4 weeks of age, the genotype ofmales was determined by PCR of tail biopsy DNA.Homozygotic transgenic males were randomized intofour groups: (1) control diet (calories consisting of Fat25.167%, Carbohydrate 54.491%, Protein 20.341%),(2) control diet with micronutrient supplement (addedin proportion to the human equivalent of 800 IUvitamin E (a-tocopherol succinate), 200 mg of selenium(seleno-DLmethionine) and 50mg of lycopene per day),(3) a 40% calorie (cal; calories consisting of: Fat 40.11%,Carbohydrate 42.55%, Protein 17.34%) diet, and (4) 40%cal withmicronutrient supplement. This micronutrientsupplement regimen was previously reported todecrease incidence of prostatic malignancy in Lady(12T-10)mice on both control andhigh fat diets [40] andwas based on the standard human dosing outlined byFleshner et al. [5,42–44]. All animals were maintainedon the respective formulated diets (Purina Mills TestDiet, Richmond, Indiana) until they were 28–32 weeksof age [40]. The chowwas non-irradiated, stored at 48Cat all times, stable for 6 months (assayed by thecompany), and free of phytoestrogens. Animals wereweighed bi-weekly. Animal care and treatmentswere conducted in accordance with established guide-lines and protocols approved by the Sunnybrook andWomen’s College Health Sciences Centre, Toronto,Canada and in accordance with the Canadian Councilon Animal Care.

Preparation andAnalysis ofBloodand Tissues

Tissues collected at necropsy were routinely fixed in10% (v/v) buffered formalin. Five micrometer sections

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were cut from paraffin-embedded tissues, mounted onslides, routinely stained with hematoxylin and eosin,and processed for histopathological evaluation. Para-ffin was removed in Histoclear substitute (FischerScientific, Hampton, NH) and sections were re-hydrated in 100% EtOH, 95% EtOH, 70% EtOH, andPBS baths, respectively. Sections were treated with 1%Triton-X to permeabilise cells, followed by heatactivated antigen retrieval in 0.1 M Citrate buffer for30 min. Endogenous peroxidase activity was neutral-ized in a 3% hydrogen peroxide bath. Non-specificbinding of was inhibited by an initial block using theMouse-on-mouse (MOM) kit (Vector labs, Burlingame,CA) according to manufacturer’s specifications,washed once in PBS for 10 min and then incubatedwith CAS block (Zymed/Invitrogen, Burlington, ON,Canada) for 10 min at room temperature.

Antibody Preparation,Incubationand Recognition

Antibodies were optimized on cell lines/tissuesections and diluted in CAS block accordingly. Anti-bombesin (Immunostar, Hudson, WI) was diluted1:2,500, anti-CgA (Chemicon, Temecula, CA) wasdiluted 1:250, anti-calcitonin (Research Diagnostics,Concord, MA) was diluted 1:200, anti-NSE (Dako) wasdiluted 1:1,000, anti-PTHrP (Santa Cruz, Santa Cruz,CA) was diluted 1:50, anti-neurotensin (Santa Cruz)was diluted 1:50 and anti-serotonin (Abcam, Cam-bridge, UK)was diluted 1:200. Sections were incubatedin a humidified chamber at 48C overnight. Sectionswere washed three times in PBS for 5min then incubat-ed with the ABC kit (Vector labs Universal secondaryantibody) according to manufacturers recommenda-tions. Sections were washed three times in PBS andbinding was visualized following incubation withVector DAB. Sections were washed in water, counterstainedwithhematoxylin andHarleco StainingBlueingReagent (EM Sciences, Kansas City, MO), dehydratedand mounted in non-aqueous mounting media.

Quantitation of Staining andDataAnalysis

Sections were photographed using the Zeiss Axio-plan2 (Oberkochen, Germany) in phase contrast at200�magnification.When possible, 10 random imageswere captured and stored until analysis. Stained cellswere counted post-image capture using the manualcount function on theNorthern Eclipse image software.Data were compiled, sorted into groups and comparedstatistically using SPSS statistical analysis software.An unpaired, two sided Student’s t-test was used tocompare treatment groups with the assumption ofunequal variance.

RESULTS

AHigh Fat Diet PromotesCgAand Serotonin Expression

Prostatic tissue obtained from five animals from thecontrol and high-fat dietary groups were compared forexpression of all NEmarkers in prostate epithelial cells(Fig. 1A). The number of epithelial cells positive forCgA and serotonin were significantly increased intissue from animals on a high-fat diet (Fig. 1A,B). CgA-positive cell fraction increased over 10-fold whileserotonin-positive cell fraction increased at least four-fold. SV40 large Twas not altered between groups [40],which confirms both an increase in tumor burden andpossibly the glycolytic state of the tumor.

Micronutrients DecreaseTumorBurden andAlteredNSE,PTHrP, andBombesin Expression

It has been previously demonstrated that theadministration of micronutrients to immature Lady(12T-10) transgenic male mice inhibited the develop-ment of PCa and prolonged the disease-free time forthese animals [40]. The effect of treatment with micro-nutrients on NE marker expression was comparedwithin each dietary group (Fig. 2A). Animals fed acontrol diet were compared with animals placed on acontrol diet supplemented with micronutrients fromweaning. A similar comparison was made betweenhigh-fat diet fed animals and matched diet plusmicronutrient animals. A significant effect of micro-nutrient treatment was apparent in the morphology ofthe prostates and the expression of NSE, PTHrP, andbombesin in both diet groups (Fig. 2B). As previouslydescribed, the transformed phenotype was signifi-cantly reduced in mice receiving the micronutrientsupplement. When compared for expression of the NEmarker panel, prostate tissue from micronutrient-supplemented diets exhibited significantly increasedthe number of PTHrP- and bombesin-positive cells,while NSE expression was significantly decreasedindependent of diet.

AHigh FatDietMaintains Signif|cantNEMarkerExpression in the Presence ofMicronutrients

A comparison between the prostates of animals fedmicronutrients on the control diet or high-fat dietindicated that micronutrient supplementation did notblock expression of the NE marker CgA (Fig. 2A).However, expressionof calcitonin andneurotensinwassignificantly suppressed in control diet animals whilenot affected in high fat diet animals. Therefore, theability of micronutrient supplementation to suppressphenotypic transformation in control diet animals was

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correlated with decreased expression of CgA, NSE,calcitonin, and neurotensin., while in high fat dietanimals was only correlated with decreased NSEexpression (Fig. 3A,B).

DISCUSSION

Anormal population of NE cells exists in the diseasefree prostate. The Lady (12T-10) transgenic model has

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Fig. 1. CgAandSerotoninaresignificantlyelevatedbyhighfatintakeinLady (12T-10)Transgenicmice.A:Animalswere fedeither acontrolorhigh fatdiet (40%cal) fromweaning.Prostate tumorswere takenfromtheanimals at approximately30weeksof age. Stainingof allNEmarkershighlighteda significantelevationofCgApositive (P¼ 0.0003) andSerotoninpositive (P¼ 0.0003) cells in thehigh fatdiet.Barrepresentsmeannumber of positive (þve) epithelial cells per 200� phase field.Error bars represent standard error of themean, **P< 0.01.B: Representativephotomicrographsofimmunocytochemicalstaining forCgAandSerotoninintheLady (12T-10)Transgenicmouseprostateofanimals fedacon-troldietorhigh fatdiet.Imageswerecapturedusinga20�objectivewith200� totalmagnification.

348 Palmeret al.

been demonstrated to have significant NE positiveprostatic carcinoma, as determined by CgA expression[11]. Development of advanced disease is significantlyincreased in animals maintained on a high fat diet anddecreased by micronutrient supplementation [40]. Weexamined sevendifferentNEmarkerswith significancein relation to disease progression, diet and micro-nutrient supplementation. Expression of CgA, whichhas growing clinical significance in human patients asanalternativemarker for non-PSAreactive tumors,waselevated further by high fat diet, independent ofmicronutrient treatment. This supports the importanceof CgA as a potential marker of disease progressionin transformed cells. Serotonin expression, which hasbeen suggested to affect mitogenesis and migration ofPCa models in vitro [31,32], was also elevated in

prostates of high fat diet animals. Curiously, whilemicronutrient supplementation had no effect onserotonin expression in high fat diet animals, itsexpression was elevated approximately fivefold bymicronutrient supplementation in control diet animals.The Lady (12T-10) model may provide an excellentplatform in which to determine how serotonin mightinfluence mitotic or metastatic potential of PCa in anin vivo system.

Since micronutrient supplementation is correlatedwith decreased disease burden, we analyzed how suchsupplementation of diets might influence the profileof NE marker expression. We found that independentof diet, the proportion of cells that stained positive forPTHrP and bombesin, were elevated in the prostatesof animals on micronutrients. As PTHrP has been

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Fig. 2. Micronutrients increase PTHrP and Bombesin expression independent of diet in Lady (12T-10) transgenic mouse prostates.A: Prostate sections of animals fedeither a controldietorhigh-fatdiet (40%cal)were comparedwith those fedmicronutrients fromweaninguntilapproximately30weeksofage.StainingofPTHrPandBombesinwaselevatedasaresultofmicronutrients(P< 0.0001andP< 0.01,respect-ively), independentof diet.Bar representsmeannumber of positive (þve) epithelial cells per 200� phase field.Error bars represent standarderrorof themean,*P< 0.05,**P< 0.01.B:Representativephotomicrographsofimmunocytochemicalstaining forPTHrPandBombesininLady(12T-10)Transgenicmouseprostateepithelia.Imageswerecapturedusinga20�objective,withmagnificationrepresentativeof200�.

Diet InfluencesNeuroendocrineDifferentiation 349

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Fig. 3. Micronutrients do not block the expression of CgA,NSE,Calcitonin and Neurotensin in the high fat diet.A: Prostate sections ofanimals fedmicronutrients oneither acontrolorhigh fatdiet (40%cal) for approximately30weekswerecompared forNEmarkerexpression.Barrepresentsmeannumberofpositive (þve)epithelialcellsper200�phase field.Errorbarsrepresent standarderrorof themean, *P< 0.05,**P< 0.01. B: Representative photomicrographs of immunocytochemical staining for CgA, NSE, Calcitonin and Neurotensin in the Lady(12T-10)transgenicmouseprostateofanimalsreceivingmicronutrienttreatmentoneitheracontrolorhighfatdiet.Imageswerecapturedusinga20�objective,withmagnificationrepresentativeof200�.

350 Palmeret al.

detected in the serum of patients with hypercalcemia,elevations in this peptidemay be important in the earlyestablishment of bone metastases. Mounting evidencepoints to the use of bisphosphonates as palliativeor preventative therapies, suggesting pre-treatmentwhich inhibits bone loss may also prevent bonemetastasis or skeletal events [45,46]. In addition, thiselevation may be related to the potentiality of theepithelial cells in the Lady (12T-10) transgenic prostates,where the cells may still have the ability to transformonce the ‘‘inhibitor’’ is removed. As the epithelial cellsof the Lady (12T-10) transgenic maintain expression ofthe Large T antigen on a control, high fat or micro-nutrient supplemented diet, the oncogenic potential ofthe antigen is still present yet may be suppressed bymicronutrient treatment. The expression of bombesinby epithelial cells in a micronutrient treated prostate,which has relatively normal morphology, may point toits role as an early marker of transformation andoncogenicity, mainly due to its mitogenic affects inPCa cell lines [47]. Micronutrients may suppress themitogenic affect of bombesin enough to maintain a‘‘normal phenotype,’’ however if any further trans-formation occurred this inhibition may be lost. Thiswould account for the paradoxical observation ofdecreased tumor burden in the face of elevated NEDas such NE transdifferentiated cells have themselvesbeen established to exhibit lower basal mitotic rates[48,49]. As PCa has been reported to have significantand varied chromosomal abnormalities these datastrengthen the argument for multiple markers atdiagnosis and combination treatments [50]. Futurework will examine whether tumor development isaltered after withdrawal of micronutrients.

Interestingly, NSE, a common NE marker, wasdecreased in the micronutrient treated prostates ascomparedwith their untreated controls. This points to apossible loss of glycolytic state, as the tumor burdenwas significantly decreased and the gland wasadequately perfused. Comparison of animals with‘‘normal’’ prostate phenotype following micronutrienttreatment on either the control or high fat diethighlighted a significant elevation of CgA, NSE,calcitonin and neurotensin. While CgA and NSE haveclassically been used as markers of NE differentiation,the use of additional markers such as calcitonin andneurotensinmay provide information as to therapeuticoptions. Calcitonin’s ability to inhibit chemotherapeu-tic apoptosis in cancer cell lines [51] points to therequirement of alternatives or adjuncts to chemo-therapy in treatment regimes. As neurotensin may beinvolved in the autocrine growth of PCa cells, it hasbeen suggested that simply targeting the neuropeptidereceptorsmay be an alternative therapywith decreasedsystemic impact [52].

Thus, the Lady (12T-10) transgenic model providesinsight into the putative benefits of micronutrientsupplementation in prevention of disease develop-ment. Further analysis of the interaction of diet,chemotherapeutic agents and micronutrients in theLady (12T-10) transgenic will further our understand-ing of the role of NE differentiation in the advancementof prostate disease.

ACKNOWLEDGMENTS

We thank Crocetta Accardi (Comparative Research)for excellent technical assistance.

REFERENCES

1. Jemal A, Siegel R, Ward E, Murray T, Xu J, Thun MJ. Cancerstatistics, 2007. CA Cancer J Clin 2007;57(1):43–66.

2. Bostwick DG, BurkeHB, DjakiewD, Euling S, Ho SM, LandolphJ, Morrison H, Sonawane B, Shifflett T, Waters DJ, Timms B.Human prostate cancer risk factors. Cancer 2004;101 (10 Suppl):2371–2490.

3. RubinMA,DeMarzoAM.Molecular genetics of humanprostatecancer. Mod Pathol 2004;17(3):380–388.

4. Fleshner N, Bagnell PS, Klotz L, Venkateswaran V. Dietary fatand prostate cancer. J Urol 2004;171(2 Pt 2):S19–S24.

5. FleshnerNE.VitaminE andprostate cancer.UrolClinNorthAm2002;29(1):107–113, ix.

6. Clinical Guidelines on the Identification, Evaluation, and Treat-ment of Overweight and Obesity in Adults–The EvidenceReport. National Institutes of Health. Obes Res 1998;6 (Suppl 2):51S–209S.

7. Fleshner N, Al Azab R. Prostate cancer: Chemopreventionupdate 2005. Can J Urol 2005;12 (Suppl 2):2–4.

8. Fleshner N, Fair WR, Huryk R, Heston WD. Vitamin E inhibitsthe high-fat diet promoted growth of established humanprostate LNCaP tumors in nude mice. J Urol 1999;161(5):1651–1654.

9. Fleshner NE, Kucuk O. Antioxidant dietary supplements:Rationale and current status as chemopreventive agents forprostate cancer. Urology 2001;57 (4 Suppl 1):90–94.

10. Gingrich JR, Barrios RJ, Morton RA, Boyce BF, DeMayo FJ,Finegold MJ, Angelopoulou R, Rosen JM, Greenberg NM.Metastatic prostate cancer in a transgenic mouse. Cancer Res1996;56(18):4096–4102.

11. MasumoriN, ThomasTZ,ChaurandP,CaseT, PaulM,Kasper S,CaprioliRM,TsukamotoT, Shappell SB,MatusikRJ.Aprobasin-large T antigen transgenic mouse line develops prostateadenocarcinomaandneuroendocrine carcinomawithmetastaticpotential. Cancer Res 2001;61(5):2239–2249.

12. Amorino GP, Parsons SJ. Neuroendocrine cells in prostatecancer. Crit Rev Eukaryot Gene Expr 2004;14(4):287–300.

13. Krijnen JL, Bogdanowicz JF, Seldenrijk CA, Mulder PG, vander Kwast TH. The prognostic value of neuroendocrinedifferentiation in adenocarcinoma of the prostate in relation toprogression of disease after endocrine therapy. J Urol 1997;158(1):171–174.

14. di Sant’Agnese PA. Neuroendocrine differentiation in humanprostatic carcinoma. Hum Pathol 1992;23(3):287–296.

The Prostate

Diet InfluencesNeuroendocrineDifferentiation 351

15. Guate JL, Escaf S, Menendez CL, del Valle M, Vega JA.Neuroendocrine cells in benign prostatic hyperplasia andprostatic carcinoma: Effect of hormonal treatment. Urol Int1997;59(3):149–153.

16. Bonkhoff H, Remberger K. Differentiation pathways andhistogenetic aspects of normal and abnormal prostatic growth:A stem cell model. Prostate 1996;28(2):98–106.

17. Bonkhoff H, Wernert N, Dhom G, Remberger K. Relation ofendocrine-paracrine cells to cell proliferation in normal, hyper-plastic, and neoplastic human prostate. Prostate 1991;19(2):91–98.

18. Abrahamsson PA. Neuroendocrine differentiation and hor-mone-refractory prostate cancer. Prostate Suppl 1996;6:3–8.

19. Pruneri G, Galli S, Rossi RS, Roncalli M, Coggi G, Ferrari A,Simonato A, Siccardi AG, Carboni N, Buffa R. Chromogranin Aand B and secretogranin II in prostatic adenocarcinomas:Neuroendocrine expression in patients untreated and treatedwith androgen deprivation therapy. Prostate 1998;34(2):113–120.

20. Wafa LA, Palmer J, Fazli L, Hurtado-Coll A, Bell RH,NelsonCC,Gleave ME, Cox ME, Rennie PS. Comprehensive expressionanalysis of L-dopa decarboxylase and established neuroendo-crine markers in neoadjuvant hormone-treated versus varyingGleason grade prostate tumors. Hum Pathol 2007;38(1):161–170.

21. Dauge MC, Delmas V. A.P.U.D. type endocrine tumour of theprostate. Incidence and prognosis in association with adenocar-cinoma. Prog Clin Biol Res 1987;243A:529–531.

22. Cohen RJ, Glezerson G, Haffejee Z. Neuro-endocrine cells–anew prognostic parameter in prostate cancer. Br J Urol 1991;68(3):258–262.

23. TarleM, Frkovic-Grazio S, Kraljic I, Kovacic K. Amore objectivestaging of advanced prostate cancer–routine recognition ofmalignant endocrine structures: The assessment of serum TPS,PSA, and NSE values. Prostate 1994;24(3):143–148.

24. Theodorescu D, Broder SR, Boyd JC, Mills SE, FriersonHF Jr. Cathepsin D and chromogranin A as predictors of longterm disease specific survival after radical prostatectomy forlocalized carcinoma of the prostate. Cancer 1997;80(11):2109–2119.

25. BostwickDG,Qian J, Pacelli A, ZinckeH, BluteM, Bergstralh EJ,Slezak JM, Cheng L. Neuroendocrine expression in nodepositive prostate cancer: Correlation with systemic progressionand patient survival. J Urol 2002;168(3):1204–1211.

26. Hvamstad T, Jordal A, Hekmat N, Paus E, Fossa SD. Neuro-endocrine serum tumourmarkers in hormone-resistant prostatecancer. Eur Urol 2003;44(2):215–221.

27. Cussenot O, Villette JM, Cochand-Priollet B, Berthon P. Evalua-tion and clinical value of neuroendocrine differentiation inhuman prostatic tumors. Prostate Suppl 1998;8:43–51.

28. Deftos LJ, Abrahamsson PA. Granins and prostate cancer.Urology 1998;51 (5A Suppl):141–145.

29. Deftos LJ, Nakada S, Burton DW, di Sant’Agnese PA, CockettAT, Abrahamsson PA. Immunoassay and immunohistologystudies of chromogranin A as a neuroendocrine marker inpatients with carcinoma of the prostate. Urology 1996;48(1):58–62.

30. Feldman SA, Eiden LE. The chromogranins: Their rolesin secretion from neuroendocrine cells and as markers forneuroendocrine neoplasia. Endocr Pathol 2003;14(1):3–23.

31. Lee LF, Guan J, Qiu Y, Kung HJ. Neuropeptide-inducedandrogen independence in prostate cancer cells: Roles of

nonreceptor tyrosine kinases Etk/Bmx, Src, and focal adhesionkinase. Mol Cell Biol 2001;21(24):8385–8397.

32. Elek J, Pinzon W, Park KH, Narayanan R. Relevant genomicsof neurotensin receptor in cancer. Anticancer Res 2000;20(1A):53–58.

33. Motellon JL, Javort Jimenez F, de Miguel F, Jaras MJ, Diaz A,Hurtado J, Esbrit P. Parathyroid hormone-related protein,parathyroid hormone, and vitamin D in hypercalcemia ofmalignancy. Clin Chim Acta 2000;290(2):189–197.

34. Boabaid F, Berry JE, Koh AJ, SomermanMJ, McCcauley LK. Therole of parathyroid hormone-related protein in the regulation ofosteoclastogenesis by cementoblasts. J Periodontol 2004;75(9):1247–1254.

35. Matsuzaki K, Udagawa N, Takahashi N, Yamaguchi K, YasudaH, ShimaN,MorinagaT, ToyamaY, YabeY,HigashioK, Suda T.Osteoclast differentiation factor (ODF) induces osteoclast-likecell formation in human peripheral blood mononuclear cellcultures. Biochem Biophys Res Commun 1998;246(1):199–204.

36. Evangelou AI, Winter SF, Huss WJ, Bok RA, Greenberg NM.Steroid hormones, polypeptide growth factors, hormone refrac-tory prostate cancer, and the neuroendocrine phenotype. J CellBiochem 2004;91(4):671–683.

37. Deeble PD, Murphy DJ, Parsons SJ, Cox ME. Interleukin-6- andcyclic AMP-mediated signaling potentiates neuroendocrinedifferentiation of LNCaP prostate tumor cells. Mol Cell Biol2001;21(24):8471–8482.

38. Farini D, Puglianiello A, Mammi C, Siracusa G, Moretti C. Dualeffect of pituitary adenylate cyclase activating polypeptide onprostate tumor LNCaP cells: Short- and long-term exposureaffect proliferation and neuroendocrine differentiation. Endo-crinology 2003;144(4):1631–1643.

39. Deeble PD, Cox ME, Frierson HF Jr, Sikes RA, Palmer JB,Davidson RJ, Casarez EV, Amorino GP, Parsons SJ. Androgen-independent growth and tumorigenesis of prostate cancer cellsare enhanced by the presence of PKA-differentiated neuro-endocrine cells. Cancer Res 2007;67(8):3663–3672.

40. Venkateswaran V, Fleshner NE, Sugar LM, Klotz LH. Antiox-idants block prostate cancer in lady transgenic mice. Cancer Res2004;64(16):5891–5896.

41. Greenberg NM, DeMayo F, Finegold MJ, Medina D, Tilley WD,Aspinall JO, Cunha GR, Donjacour AA, Matusik RJ, Rosen JM.Prostate cancer in a transgenic mouse. Proc Natl Acad Sci USA1995;92(8):3439–3443.

42. Venkateswaran V, Fleshner NE, Klotz LH. Synergistic effect ofvitamin E and selenium in human prostate cancer cell lines.Prostate Cancer Prostatic Dis 2004;7(1):54–56.

43. Venkateswaran V, Fleshner NE, Klotz LH. Modulation of cellproliferation and cell cycle regulators by vitamin E in humanprostate carcinoma cell lines. J Urol 2002;168(4 Pt 1):1578–1582.

44. VenkateswaranV,Klotz LH, FleshnerNE. Seleniummodulationof cell proliferation and cell cycle biomarkers in human prostatecarcinoma cell lines. Cancer Res 2002;62(9):2540–2545.

45. Clark PE, Torti FM. Prostate cancer and bone metastases:Medical treatment. Clin Orthop Relat Res 2003; (415 Suppl):S148–S157.

46. Gilbert SM, McKiernan JM. Epidemiology of male osteoporosisand prostate cancer. Curr Opin Urol 2005;15(1):23–27.

47. Yegen BC. Bombesin-like peptides: Candidates as diagnosticand therapeutic tools. Curr Pharm Des 2003;9(12):1013–1022.

48. Palmer J, Ernst M, Hammacher A, Hertzog PJ. Constitutiveactivation of gp130 leads to neuroendocrine differentiationin vitro and in vivo. Prostate 2005;62(3):282–289.

The Prostate

352 Palmeret al.

49. Cox ME, Deeble PD, Lakhani S, Parsons SJ. Acquisition ofneuroendocrine characteristics by prostate tumor cells isreversible: Implications for prostate cancer progression. CancerRes 1999;59(15):3821–3830.

50. Konishi N, Shimada K, Ishida E, Nakamura M. Molecularpathology of prostate cancer. Pathol Int 2005;55(9):531–539.

51. Vilches J, Salido M, Fernandez-Segura E, Roomans GM. Neuro-peptides, apoptosis and ion changes in prostate cancer.Methodsof study and recent developments. Histol Histopathol 2004;19(3):951–961.

52. Reubi JC, Macke HR, Krenning EP. Candidates for peptidereceptor radiotherapy today and in the future. J Nucl Med2005;46 (Suppl 1):67S–75S.

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