Pomegranates, Polyphenols,and Prostate Cancer Prevention

Allan J. Pantuck, MD, MS, FACSAssociate Professor of Urology

David Geffen School of Medicine at UCLAFudan University

March, 2008

Revised Model of Human Prostate Cancer Pathogenesis

(DeMarzo et al)

NML

PIA

PIN

PCA

ChronicInflammation (ROS)

Prolonged oxidantand electrophile

exposureInfection?

GSTP1Inactivation

(Hypermethylation)

I II IIIa b

Androgen Dependent,Localized

Androgen Dependent,Recurrent or

Metastatic

CastrationResistant,Metastatic

GSTs,MEHNQO1,UGTs

P450Enzymes

Cancer

ExcretionDetoxifiedmutagens

MutagensProcarcinogens

Pomegranate Anthocyanins, Isoflavones,Phytoestrogens, Catechins

Hypothesis:Gene-Nutrient Interactions May Modify

Development/Growth of Prostate Cancer

DNAdamage

Adapted from HJ Lin et al, CEBP 7:647, 1998

LegendPromote

Inhibit

Agents with ChemopreventivePotential for Prostate Cancer

Anti-estrogens[ER-alpha/beta, Aromatase, TGFalpha](Toremifene, Raloxifene, Arimidex, I3C/DIM)

Differentiation Agents[VDR, RXR, HDAC, DNMT](Vit D Analogs, Targretin, SAHA, valproic acid)

Anti-proliferation/Cell Cycle Agents[ODC, RXR/RAR, p21,p27](DFMO, Panretin, Vitamin D)

Signal Transduction Modulators (Kinases)[EGFR (Tarceva), PDGFR (STI-571),VEGFR/PDGFR (SU-11248), PI3K/AKT/mTOR (Celecoxib, Rapamycin), NFkB (polyphenols)

Ras/Farnesyl/Geranyl TransferaseModulators(Statins, SCH 66336, L-778,123,Perillyl Alcohol)

Anti-angiogenesis Modulators(Thalidomide analogs, Avastin, Celecoxib)

PPAR (gamma/delta) Modulators(Glitazones, DHEA Analogs, Retinoids, NSAIDs, 15d-PGJ2)

IGF-1/IGFBP-3 Modulators(Soy, Retinoids,Tamoxifen, Lycopene, Vit D)

Novel Growth Factor Modulators [ET-1, IL-6, NGF](Atrasentan, Celecoxib, Bowman-BirkInhibitor)

Telomerase ModulatorsAntiandrogens, SERMs, Polyphenols, Vit D, NGF

Gene-based vaccines (P53, Rb, E1A/PSA, PSMA, PSA/B7/GMCSF]

Anti-androgens[5AR-1/2, AR](Finasteride, Dutasteride, DIM, SARMs)

Antioxidant Modulators [ROS, GSH/GST-P1,iNOS](Selenium, Vitamin E, Lycopene, Polyphenols, Resveratrol, Sulforaphanes)

Arachidonic Acid Modulators[NFkB, COX (PGE2), LOX (HETE), PPARs] (ASA/NSAIDS, Celecoxib, Rofecoxib, R-flurbiprofen, NO-ASA, Vit E, Zileuton)

Phytoestrogens[ER-beta, 5AR, AR, RTK](Genistein, Daidzein/Equol, Resveratrol)

Phenolics in Pomegranate Juice

*Phenolics include phenols, which have one phenol unit in their structure, and polyphenols, which have >1 phenol unit in their structure. For simplicity, we call all the compounds Polyphenols.

DelphinidinCyanidinPelargonidin• 10% of total polyphenols• Potent antioxidant• Pigments – responsible for

color of pomegranate juice• Same antioxidant/

pigments in blueberry and cherry

Galloyl glucose• Unknown % of totalpolyphenols

• Potent antioxidant

Punicalagin• Majority of totalpolyphenols

• Highly potentantioxidant

• Pomegranate isrichest food source

• Breaks down to giveellagic acid

Resveratrol• Main active ingredient in

red wine• Pom juice has 1/10 the

amount as red wine

Coumestrol• Also termed a

phytoestrogen• Same type of

beneficial compounds as in soy

Ellagic acid• Unknown percent of

total polyphenols• Potent antioxidant • Pomegranate is richest

food source• Also found in

strawberry and raspberry

• Some evidence for anti- cancer effects

Gallic acid• Unknown % of total

polyphenols• Potent antioxidant • Unknown health

effects

Phenolics*

Polyphenols Phenols

Hydrolyzable Tannins Stilbenes Flavonoids

Ellagitannins Gallotannins Anthocyanins Isoflavones

Pomegranates and Prostate CancerPre-Clinical Data Summary: 2001

In vitro studies show:

• 59-75% growth inhibition of PC3• Delayed progression into S phase• Induction of apoptosis

In vivo studies in SCID mice show:

• 52% growth inhibition of LAPC-9 tumors• 70% reduction in PSA• Prolonged survival

Inhibition of In vivo Orthotopic Prostate Tumor Growth by Pomegranate Juice

Tumor Weight at day 46 PSA Levels

0.000.200.400.600.801.001.201.401.601.802.00

Tum

or W

eigh

t (g)

Weights(g)

1.13 0.64

Control PJC Treated

0.0

50.0

100.0

150.0

200.0

250.0

300.0

DaysP

SA

Blo

od L

evel

s (n

g/m

l)

Control 8.3 43.2 111.2 106.4 146.8 182.3

PJC Treated 1.1 41.9 45.1 53.3 47.0 60.1

13 20 27 34 41 46

**

♦ Treatment with PJC significantly reduced tumor growth of LAPC-9 xenograftsas measured by tumor weight (55%) and PSA levels (70%), as compared to control group (7 mice per group).

*p ≤ 0.05

Experiment 1

Phase II Study for Men with Rising PSAAfter RRP or XRT: 2002

• Men with Recurrent Prostate Cancer• Rising PSA after RRP or XRT• Low risk: PSA < 5, Gleason < 8• No evidence of metastatic disease• No previous hormone therapy• Baseline PSA DT

Pantuck et alClinical Cancer Research, 2006

Treatment

8 oz. Pomegranate juice dailyproviding 1.5 mmol polyphenols

I II IIIa b

PSADT Before Baseline

PSADT After Baseline

Change

Mean ± SD 15.0 ± 11.1 54.0 ± 53 39.00 ± 45.6

Signed Rank Test

P=0.0001

“Final” Results 2005: PSA Doubling TimeYear 3

Pantuck et alClinical Cancer Research, 2006

PSADT Before Baseline

PSADT After Baseline

Change

Mean ± SD 15.0 months 58 months 43.0 months

P=0.0001

Updated Results 2007: PSA Doubling TimeYear 5

Non-Active 51 months

Active 69 months

Effect on PSA Doubling Time

-3

-2

-1

0

1

2

3

-60 -50 -40 -30 -20 -10 0 10 20 30 40 50 60

Log

PSA

median rate pre

Months follow up (0=treatment start)

median rate post

In Vitro Apoptosis:Effect of Patient Serum on LnCaP

Apoptosis Data

0.0000.0200.0400.0600.0800.1000.1200.140

1 3 5 7 9 11 13 15 17 19 21 23 25 27 29 31 33 35 37 39 41 43 45 47 49 51 53

Screen ID#

Apoptosis 9 month Apoptosis Baseline

Apoptosis Data - Percent Change

-100.000-50.000

0.00050.000

100.000150.000200.000250.000300.000

1 3 5 7 9 11 13 15 17 19 21 23 25 27 29 31 33 35 37 39 41 43 45 47 49 51 53

Screen ID#

Apoptosis % change

Average Increase in Apoptosis Over Baseline= 17.5% p=0.0004

Updated Results

Approx. 30% remain on study with stable disease

35% achieved decreased PSA (range 5%-85%)

82.5% had improved PSA doubling time

Nearly 4-fold prolongation of PSA DT

Effect on PSA DT appears to be durable

Significantly decreased proliferation, increased apoptosis in in vitro mitogenic assays

Delaying Time to Hormone Therapy(a relevant time point for prostate cancer)

Treated with chemopreventive agents

0%

100%

Time

PSA

ng/

ml

Need For HormoneTherapy

AACR Task Force“Patient Benefit by Reducing Cancer Risk and/or by

Decreasing the Need for Invasive Interventions”Clin Ca Res 8: 314-346, 2002

Translational Research

2007 Prostate CancerPomegranate Research Portfolio

• POM Phase II Continuation Study• POM Phase III Multi-Center Study• Neoadjuvant POM Pill Study

• POM and NFκB Pathway• POM and IGF Axis

• Cleveland Clinic Foundation, Cleveland, OH• Columbia University, New York, NY

• Mayo Clinic, Rochester, MN• MD Anderson Cancer Center, Houston, TX• University of California, Los Angeles, CA

• University of California, San Francisco, CA

Phase III Study Sites

2007 Prostate CancerPomegranate Research Portfolio

• POM Phase II Continuation Study• POM Phase III Multi-Center Study• Neoadjuvant POM Pill Study

• POM and NFκB Pathway• POM and IGF Axis

Polyphenols May Affect Proliferation Via NFκB

PE inhibits constitutive and TNFα-induced NF-κB activity

PE inhibits constitutive NF-κB activity in a dose-dependent fashion

0

0.2

0.4

0.6

0.8

1

1.2

1.4

0 3000 2000 1000

Dilution Factor

RLU

CL1DU145LAPC4LNCaP-AR

PE inhibits constitutive and TNFα-induced NF-κB (EMSA)

DU145

NF-κB

TNFα - - - - - + + + + + - -

Cold competition

0

1:32

00

1:16

00

1:80

0

1:40

0 01:

3200

1:16

00

1:80

0

1:40

0 0 0

wt mut

Oct-1

NF-κB

TNFα - - - - - + + + + + - -

Cold competition

0

1:32

00

1:16

00

1:80

0

1:40

0 01:

3200

1:16

00

1:80

0

1:40

0 0 00

1:32

00

1:16

00

1:80

0

1:40

0 01:

3200

1:16

00

1:80

0

1:40

0 0 0

wt mut

Oct-1

PE inhibits constitutive and TNFα-induced NF-κB (EMSA) cont.

CL1

1:30

00

1:20

00

1:10

00

1:50

0

TNFα − + + + + + + +

00 00

NF-κB

Oct-11:

3000

1:20

00

1:10

00

1:50

0

TNFα − + + + + + + +

00 00

NF-κB

Oct-1

TNFα − + + + + + + +

00 00

NF-κB

Oct-1

Cold competitionwt mut

CL1

DU145

NF-κB

NF-κB

TNFα − + + +POM EX (1:1600) 0 0 2h 4h

Exposure of CLI and DU145 to PE led to a time-dependent

decrease in NFκB levels

Exposure of DU145 and LAPC4 cells to PE led to a time-dependent increase in IκBα levels (Western)

TNFα - + + + + - + + + + + + +

PE exposure (hr) 0 0 2 4 8 0 0 2 4 8 2 4 8

PE concentration 1:1600 1:1600 1:800 DU145 LAPC4

IkBα

actin

TNFα - + + + + - + + + + + + +

PE exposure (hr) 0 0 2 4 8 0 0 2 4 8 2 4 8

PE concentration 1:1600 1:1600 1:800 DU145 LAPC4

IkBα

actin

0

5

10

15

20

25

0 20000 10000 5000 2500 1250

PomEX Dilution Factor (12h)

hIL6

(pg/

mL)

hIL6(pg/mL)

PE inhibits IL-6 protein

PE inhibits prostate cancer growth in vitro

PE more effectively inhibited overall growth of DU145 cells than

did the pomegranate juice

0

0.2

0.4

0.6

0.8

1

1.2

Veh 2000 1000 800 600 500 400 300 200 100 50Dilution factor

Rel

ativ

e ce

ll vi

abili

ty PJPE

PE more effectively inhibited overall growth of DU145 cells than did the pomegranate juice (cont.)

PE, 1:100

Veh

PE, 1:400

PE, 1:1000

PE inhibits expansion of prostate cancer cell lines

0

0.2

0.4

0.6

0.8

1

1.2

Veh 8000 4000 2000 1000 500

Dilution factor

Rel

ativ

e ce

ll vi

abilit

y

CL1LAPC4LNCaP-AR

Cell growth inhibition is attributable to inhibition of apoptosis (Annexin V)

Dilution Apoptosis (%)

Veh 11.1

1:4000 20.6

1:2000 31.4

1:1000 60.7

1:500 67.4

Veh

PE (1:2000)

0

10

20

30

40

50

60

70

80

90

100

0 2x103 1x103 500

POM Dilution

% A

popt

osis

Controlp65/p50

Inhibition of NF-κB activation by PE is required for maximal apoptosis induced by PE

Cell growth inhibition may also be attributable to inhibition of proliferation

Dilution G1 (%)

S (%)

Veh 64.1 34.3

1:4000 60.0 37.7

1:2000 58.8 39.2

1:1000 70.8 26.7

1:500 72.7 19.6

Veh

PE (1:1000)

PE inhibits prostate cancer growth in vivo

PE inhibits growth of androgen-independent LAPC4 xenografts

0

0.1

0.2

0.3

0.4

0.5

0.6

Inoculation Treatment Castration Week 1 Week 2

Tum

or V

olum

e (c

c)

PEVeh

PE diet reduces proliferation (Ki67)

Veh PE

PE increases apoptosis (TUNEL staining)

Veh PE

PE inhibits NF-κB in androgen-independent LAPC4 xenografts

Mouse 3 4 6 11 1 2 3 4 17L 17T 20T 20N

Castrated IntactPOMPBS PBS

NF-κB

Oct-1

Lamin B

A

p-IκBα

actin

B POMPBSMouse 3 4 6 11 1 2 3 4

Castrated

Hormone Refractory LAPC-4In Vivo Murine Model

I II IIIa b

Pom Pill Neoadjuvant Phase II Study

Protocol to begin 3Q 08—UCLA and Johns Hopkins

POM-X Pills

Placebo controlled

1 month adjuvant treatment post-radical prostatectomy

70 patients

Endpoints: biomarkers (8oxoguanine, inflammation,Apoptosis, IGF, NFKb, etc)

I II IIIa b

2007 Prostate CancerPomegranate Research Portfolio

• POM Phase II Continuation Study• POM Phase III Multi-Center Study• Neoadjuvant POM Pill Study

• POM and NFκB Pathway• POM and IGF Axis

The GH-IGF-IGFBP axis

Transport

CellularIGFBP

Receptors RXR-Nuclear actions

NuclearReceptors

Cell surfaceBP-receptors

---IGFBPs

TypeIGF Receptor

Iα

β

Survival &MitogenesisVia IRS-1 --Akt, MAPK and other pathways

IGF-I

IGF-II

Insulin

PC

Differentiation growth, aging, metabolism & carcinogenesis

ALS

150 kDa complex

IGFBP-1 IGFBP-2 IGFBP-3 IGFBP-4 IGFBP-5 IGFBP-6

TypeIGF Receptor

II

IGF2 binding

Mannose-6-phosphate bindingRetinoid binding

Growthinhibition

POM increases IGFBP-1 in humans

� C ontrol Pom0

15

30 *

Synergism of IGFBP-3 and Pom-X in LAPC4 in vitro

0

0.5

1

1.5

2

2.5

3

3.5

IGFBP-3 - + - +

PomX - - + +

**

**

**

apop

tosi

s

The Future: Prostate Cancer Prevention

I II IIIa b

AcknowledgementsUCLA UrologyWilliam Aronson, MDArie Belldegrun, MDAllan Pantuck, MDNazy Zomorodian, MS

UCLA MedicineEd Barnard, MDPinchas Cohen, MDDavid Heber, MD, PhDLouis Ignarro, PhDMatthew Rettig, MDNavindra Seeram, PhDTechnion Institute

Michael Aviram, PhD

UCLA BiomathematicsRobert Elashoff, PhDJeffrey Gornbein, PhDHejing Wang, MD

Pom WonderfulSense Foundation