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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
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
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 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 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
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
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)
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
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
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