Published OnlineFirst August 28, 2013; DOI: 10.1158/1940-6207.CAPR-12-0423
A Double-Blind, Randomized, Neoadjuvant Study of the Tissue
Effects of POMx Pills in Men with Prostate Cancer Before Radical
Prostatectomy
Stephen J. Freedland, Michael Carducci, Nils Kroeger, et al.
Cancer Prev Res 2013;6:1120-1127. Published OnlineFirst August 28, 2013.
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Published OnlineFirst August 28, 2013; DOI: 10.1158/1940-6207.CAPR-12-0423
Cancer
Prevention
Research
Research Article
A Double-Blind, Randomized, Neoadjuvant Study of the
Tissue Effects of POMx Pills in Men with Prostate Cancer
Before Radical Prostatectomy
Stephen J. Freedland1,2, Michael Carducci4,5, Nils Kroeger6,10, Alan Partin4,5, Jian-yu Rao7, Yusheng Jin7,
Susan Kerkoutian7, Hong Wu8, Yunfeng Li7, Patricia Creel3, Kelly Mundy3, Robin Gurganus5,
Helen Fedor5, Serina A. King4, Yanjun Zhang9, David Heber9, and Allan J. Pantuck6
Abstract
Pomegranates slow prostate cancer xenograft growth and prolong prostate-specific antigen (PSA) doubling
times in single-arm human studies. Pomegranates’ effects on human prostate tissue are understudied. We
hypothesized that orally administered pomegranate extract (POMx; Pom Wonderful) would lower tissue 8hydroxy-20 -deoxyguanosine (8-OHdG), an oxidative stress biomarker. Seventy men were randomized to two
tablets, POMx or placebo, daily up to four weeks before radical prostatectomy. Tissue was analyzed for
intraprostatic urolithin A, a pomegranate metabolite, benign and malignant 8-OHdG, and cancer pS6 kinase,
NF-kB, and Ki67. Primary endpoint was differences in 8-OHdG, and the study was powered to detect 35%
reduction. POMx was associated with 16% lower benign tissue 8-OHdG (P ¼ 0.095), which was not
statistically significant. POMx was well tolerated with no treatment-related withdrawals. There were no
differences in baseline clinicopathological features between arms. Urolithin A was detected in 21 of the 33
patients in the POMx group versus 12 of the 35 in the placebo group (P ¼ 0.031). Cancer pS6 kinase, NF-kB,
Ki67, and serum PSA changes were similar between arms. POMx before surgery results in pomegranate
metabolite accumulation in prostate tissues. Our primary endpoint in this modest-sized short-term trial was
negative. Future larger longer studies are needed to more definitively test whether POMx reduces prostate
oxidative stress, as well as further animal testing to better understand the multiple mechanisms through which
POMx may alter prostate cancer biology. Cancer Prev Res; 6(10); 1120–7. 2013 AACR.
Introduction
Given the significant morbidity associated with standard
prostate cancer treatments and the lack of U.S. Food and
Drug Administration (FDA)-approved agents for prostate
cancer prevention, there is growing interest in alternative
and complementary approaches for prostate cancer prevention and treatment (1). Pomegranate juice and its polyphenol antioxidants have been extensively studied preclinically
Authors' Affiliations: 1Department of Surgery, Durham VA Medical Center; 2Departments of Surgery (Urology) and Pathology; 3Genitourinary
Cancer Program, Duke Cancer Institute, Duke University Medical Center,
Durham, North Carolina; 4Prostate Cancer Program, Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins; 5Brady Urological Institute,
Johns Hopkins University School of Medicine, Baltimore, Maryland; 6Institute of Urologic Oncology; Departments of 7Pathology and Laboratory
Medicine, and 8Molecular and Medical Pharmacology; 9Division of Clinical
Nutrition, Department of Medicine, UCLA Medical Center, Los Angeles,
California; and 10Department of Urology, University Medicine Greifswald,
Greifswald, Germany
S.J. Freedland received salary support from the NIH, 1 K24 CA160653-01
for his efforts on this study.
Corresponding Author: Stephen J. Freedland, Duke University Medical
Center, Box 2626, Durham, NC 27710. Phone: 919-668-5946; Fax: 919668-7093; E-mail: steve.freedland@duke.edu
doi: 10.1158/1940-6207.CAPR-12-0423
2013 American Association for Cancer Research.
1120
for their in vivo and in vitro molecular effects, and clinically
for their impact on serum prostate-specific antigen (PSA)
kinetics (2).
Both in vitro and animal studies show that pomegranate
extract and pomegranate juice can inhibit prostate cancer
growth (3–8). In a single-arm human trial of men with a
rising PSA after primary therapy, pomegranate juice was
associated with statistically significant longer PSA doubling
times (PSADT) versus prestudy PSADT (i.e., more slowly
rising PSA; ref. 9). This finding was further supported by a
nonblinded randomized phase II trial of men with a rising
PSA after primary treatment randomized to 1 or 2 tablets of
daily pomegranate-X (POMx; Pom Wonderful), a pill containing concentrated pomegranate extracts (10). The study
found that 76% to 82% of men in both arms had longer onstudy PSADT values than prestudy PSADT, though there
were no differences in on-study PSADT between arms.
Given the lack of placebo control, the lack of a doseresponse, the fact a prior placebo-controlled trial found
73% of men on placebo on a similar study had longer onstudy PSADT than prestudy (11), and the lack of prostate
tissue to confirm biologic effects make interpreting these
data challenging.
Thus, we undertook a randomized, placebo-controlled
study of POMx daily for up to 4 weeks before radical
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POMx Before Radical Prostatectomy
Translational Relevance
The effect of pomegranates on human prostate tissue is
unclear. In a randomized double-blind study of men with
prostate cancer undergoing radical prostatectomy, we
found that up to 4 weeks of supplementation with the
pomegranate extract, POMx, was associated with no significant reductions in 8-hydroxy-20 -deoxyguanosine, a measure of oxidative stress. Given the presumed importance of
oxidative stress in prostate cancer development and progression, future larger longer studies are needed to more
definitively test whether POMx reduces prostate oxidative
stress, as well as further animal testing to better understand
the multiple mechanisms through which POMx may alter
prostate cancer biology.
prostatectomy. The goal was to obtain prostate tissue to
objectively measure whether pomegranate extracts were
systemically absorbed resulting in urolithin A, the predominant pomegranate metabolite, being accumulated in the
prostate, and to assess what molecular effects, if any, this
had on both benign and malignant prostate tissue biology.
Our primary outcome was the difference between arms in
prostate 8-hydroxy-20 -deoxyguanosine (8-OHdG) levels. 8OHdG is formed as the result of oxidative damage to the
DNA base 20 -deoxyguanosine and is a major product of
DNA oxidation. We choose 8-OHdG levels as our primary
outcome because oxidative damage is a key pathway in
prostate cancer development and progression (12), and
pomegranates have been shown to affect oxidative stress
(13), suggesting that altering oxidative stress may be a key
pathway through which pomegranates impact prostate
cancer biology. Moreover, 8-OHdG is considered to be a
sensitive, stable, and integral marker of oxidative damage in
cellular DNA, and is considered stable for immunohistochemistry in formalin-fixed, paraffin-embedded (FFPE)
sections, and antibodies have been widely used to evaluate
oxidative DNA damage in animal and human tissues (14).
Patients and Methods
Patients
Participants were recruited from the urology clinics at
Duke University (Durham, NC) and Johns Hopkins University (Baltimore, MD) between February 2009 to January
2011. Participants were required to have a histologic diagnosis of prostate adenocarcinoma and to be scheduled to
undergo radical prostatectomy at least 2 weeks from study
entry. The diagnostic needle biopsy was required to have at
least two cores with cancer to increase the likelihood of
having prostate cancer tissue for analysis. Subjects were
required to stop all commercially available pomegranate
products, nutritional supplements, and herbal therapies
(i.e., lycopene, vitamin E, selenium, genistein, or saw palmetto) for at least 2 weeks before starting the intervention.
Subjects were ineligible if they were currently on a 5-a reductase inhibitor, anti-androgens, or luteinizing-hormone
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releasing hormone (LHRH) agonists, or had received a
bilateral orchiectomy. The study was approved by the Institutional Review Board at each participating institution.
Study design
This was a phase II, randomized double-blind trial
designed to study intermediate biologic endpoints in serum
and tissue specimens to determine the bioavailability and
the effects on prostate inflammation, apoptosis, and proliferation of the study treatment. This trial was registered
with clinicaltrial.gov (#NCT00719030). All subjects underwent informed consent before study entry. Randomization
(1:1) was by a permuted random block design. Study
duration was up to 4 weeks, though a window of treatment
that included additional days of treatment was permitted to
accommodate standard surgical scheduling. All subjects
consumed a study-prescribed pill twice daily generally
starting on the day of randomization until the day of surgery
(last tablet the evening before surgery) but was timed to
ensure up to 4 weeks on therapy (minimum 2 weeks). For
subjects on the POMx arm, this was two POMx tablets taken
orally once daily (POM Wonderful) and for those on
placebo, it was a matching placebo pill with the same
schedule of administration (Paramount Farming). Compliance was recorded as a percentage of scheduled intakes of
study product consumed. Noncompliance was defined as
consumption of less than 80% of the scheduled intakes.
Subjects in both the groups were asked not to consume
commercially available pomegranates and to make no
additional changes to their diets during the study period.
At baseline and at the conclusion of up to 4 weeks of study
treatment, all subjects had a physical examination and
blood drawn for PSA, and whole blood for serum and
plasma. Following surgical removal and before fixation, a
1,000 mg biopsy of fresh prostate tissue was isolated. This
sample was obtained from any prostate tissue, regardless of
tumor involvement. The remainder of the prostate was fixed
in formalin and embedded in paraffin per standard processing procedures at each institution. All fresh frozen tissues
and slides cut from representative FFPE blocks were shipped
to UCLA (Los Angeles, CA) for analyses.
POMx
POMx (provided by Pom Wonderful) is a pomegranate
(Punica granatum L., Wonderful variety) fruit polyphenol
extract. POMx was developed to be used as a nutritional
supplement and has Generally Recognized as Safe status.
Each capsule contains 1,000 mg of POMx powder, which
includes up to 600 mg of polyphenol from extract, which
delivers pomegranate polyphenols in an amount equivalent
to about 8 oz of pomegranate juice. POMx powder is
produced in a two-step process: (i) extraction of polyphenols from pomegranate fruit, and (ii) purification of the
extract to produce a highly concentrated polyphenol powder. Extraction is conducted during the fruit harvest using
pressed pomegranate skins and arils with the seed completely removed. Product specifications have been established,
and batch analyses data confirm that the product is
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Freedland et al.
consistent in quality and free of microbial or chemical
contaminants. The extract has been well characterized and
contains the same compounds found in pomegranate juice,
differing only in having lower anthocyanidins and significantly higher proportional content of pomegranate polyphenols, primarily punicalagin and isomers, but the levels
in food or supplement products are limited to the amount
found in 8 oz of 100% juice.
Outcomes
The primary objective was to compare the mean differences between arms in prostatic 8-OHdG levels, a measure
of oxidative damage, in the radical prostatectomy specimen.
Secondary outcomes included between arm differences in
tissue biomarkers of prostate cancer inflammation, development and progression (NF-kB expression, pS6 kinase),
proliferation (Ki67), measurement of the pomegranate
metabolite, urolithin A, within the prostate, treatment-related toxicity, and serum PSA. Urolithin A was measured
from frozen tumor tissue without conducting frozen section
histologic analysis preventing us from knowing whether the
tissues were malignant or benign and creating only one
value of urolithin A for analyses.
Serum analysis
Serum was assayed for PSA using the standard CLIAcertified laboratories at each center as part of standard of
care.
Tissue biomarker analysis
Four micrometer thick tissue sections were cut before
staining. They were first heated to 56 C for 20 minutes,
followed by deparaffinization in xylene. The sections were
then rehydrated in graded alcohols and endogenous peroxidase was quenched with 3% hydrogen peroxide in
methanol at room temperature. The sections were then
placed in a 95 C solution of 0.01 mol/L sodium citrate
buffer (pH 7.0) for antigen retrieval. Protein blocking was
accomplished through application of 5% normal horse
serum for 30 minutes. Endogenous biotin was then blocked
with sequential application of avidin D, then biotin (A/B
blocking system). The sections were then incubated for 1
hour with various primary antibodies at room temperatures. Primary anti-8-OHdG was monoclonal antibody
purchased from (JaICA), anti-Ki67 monoclonal antibody
purchased from (DAKO), and anti-NF-kB was a polyclonal
antibody purchased from (Abcam). For anti-8-OHdG and
anti-Ki67, 1:50 dilutions were used and for anti-NF-kB,
1:300 dilutions were used. After washing, biotinylated
horse anti-mouse immunoglobulin G was applied for 30
minutes at room temperature. Next, the avidin–biotin
complex was applied for 25 minutes, and diaminobenzidine DAB (DAKO) was used as the chromagen. TBST buffer
at pH 7.4 was used for all intermediate wash steps and a
moist humidity chamber was used for prolonged incubations. The sections were counterstained with Harris’ hematoxylin, followed by dehydration and mounting. A negative
control section was prepared exactly in the same manner
1122
Cancer Prev Res; 6(10) October 2013
except omitting the primary antibody. Immunohistochemical stained slides were examined independently by a single
trained genitourinary pathologist blinded to treatment
(J.-Y. Rao). The staining intensities (graded from 0 to 3)
and percentage of staining for each staining grade were
recorded separately.
Tissue urolithin analysis
Reagents. All solvents and chemical reagents were highperformance liquid chromatography (HPLC) grade from
Fisher Scientific Co. Urolithin A was synthesized and characterized at UCLA Center for Human Nutrition. The reference mixture of urolithin A glucuronide was enriched from
human urine and characterized at UCLA Center for Human
Nutrition.
LC/MS sample preparation. Prostate samples (500
mg) were thawed and homogenized with 1.5 mL of
MeOH-HCl-H2O (79.9:0.1:20.0, v:v:v) solution using a
grinder (Kontes Duall Tissue Grinder Capacity: 5 mL size:
22 Plastic Coating). The mixture was centrifuged at 18,407
rcf for 5 minutes in a 2-mL micro centrifuge tube. The pellet
was further extracted with 1.5 mL of the same methanol
solution and centrifuged at 18,407 rcf for 5 minutes. Both
supernatants were pooled and evaporated to dryness with a
Speed-Vac. The dry residue was dissolved with 500 mL of
methanol in an ultrasound water bath for 5 minutes and
centrifuged at 18,407 rcf for 5 minutes. The resulting supernatant was evaporated to dryness. Finally, this dry residue
was reconstituted with 200 mL of MeOH: H2O (1:1) solution and centrifuged at 18,407 rcf for 5 minutes. The
supernatant was the liquid chromatography/mass spectrometry (LC/MS) sample solution.
LC/MS analyses. The LC/MS system consisted of an
LCQ Advance Finnigan system (Thermo Finnigan), equipped with a Survey HPLC system consisting of an autosampler/injector, quaternary pump, column heater, and
diode array detector with Xcalibur version 1.2 software
(Finnigan Corp). A Zorbax SB-C18 5 mm 2.1 150 mm
column (Agilent) was used for the separation with a gradient elution condition by increasing the percentage of acetonitrile (with 1% acetic acid) in water (with 1% acetic acid)
from 5% to 99% in 50 minutes at a flow rate of 0.19 mL/
minute. The MS conditions for the detection of urolithin A
glucuronide were as follows: electron spray ionization in
negative modes; scan range, 150 to 500 amu; scan rate 1
scan/second; cone voltage 17 eV. Identification of urolithin
A glucuronides was obtained by matching the molecular
ions (M-Hþ) obtained by electrospray ionization/MS and
tandem mass spectrometry (MS-MS) with the expected theoretical molecular weights from literature data as of urolithin
A glucuronide, M-H m/z 403, MS/MS (M-H m/z 227; refs. 1,
2). Subjects with an undetectable Urolithin A value were
assigned a value of 0 for statistical analyses.
Statistical analysis
A sample size of 70 (35 per group) was estimated to
provide the t test with 80% power to detect a corresponding
effect difference of 0.35 between groups with a two-sided a
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POMx Before Radical Prostatectomy
of 0.05. This power calculation was based on results from
prior interventions (15, 16). Group size was estimated using
statistical power software (Epicenter Software). No interim
analyses were planned or conducted. Continuous variables
were reported as mean (SD) and median (inter quartile
range). Normal distribution was tested with the Kolmogorov–Smirnov test. On the basis of whether the continuous data were normally distributed or not, we quantified
associations with either Student t test or the Mann–Whitney
U test, respectively. P values < 0.05 were considered significant with the Bonferroni correction applied to correct for
multiple comparisons. On the basis of normal distribution,
either Spearman or Pearson correlation was conducted to
test the association of the tumor markers with Urolithin A.
Pearson c2 and Fisher exact tests were used for comparison
of categorical variables.
ance, all other subjects in both groups were compliant with
the dietary intervention in that all men consumed more
than 80% of the prescribed pills.
There were no serious adverse events in either group. No
patient withdrew due to adverse events. Eight subjects (6
POMx, 2 placebo) reported an adverse event, all of which
were grade 1. Six of the 8 (4 POMx, 2 placebo) were gastrointestinal related (nausea, diarhhea) and judged possibly
related to study agent.
Pathologic analyses
There were no differences between groups in any pathologic endpoints (Table 2). Most men in both arms had
Gleason 7 and organ-confined, margin-negative disease.
Seminal vesicle invasion and lymph node positivity was rare.
Baseline characteristics
Though 70 subjects signed consent forms, one withdrew
before randomization. Of the remaining 69 men, 33 were
randomized to POMx and 36 to placebo. The baseline
characteristics of these 69 subjects who completed the study
are shown in Table 1. The groups were well balanced in
terms of the baseline demographics, biopsy Gleason sum,
and PSA. Most patients were White and more than 90% of
men in both arms had biopsy Gleason sums of 7 or less. All
patients had an Eastern Cooperative Oncology Group
(ECOG) score of 0.
Primary outcome
The primary outcome was between arm differences in
prostatic 8-OHdG. This was assessed both in benign prostate tissue and prostate cancer tissue. In benign tissue, 8OHdG levels were 16% lower in the POMx-treated arm,
though this failed to reach statistical significance (P ¼
0.095; Table 2). Though 8-OHdG expression in the benign
tissue was significantly correlated with the levels in cancer
tissue (r ¼ 0.441, P ¼ 0.001), the overall expression was
much lower in cancer tissue than in benign tissue (Table 2).
In cancer tissue, posttreatment 8-OHdG levels were 23%
lower in the POMx-treated arm, though this difference did
not reach statistical significance (P ¼ 0.372).
Treatment duration, compliance, and side effects
Mean number of days from screening to date of prostatectomy was 37 19 days in the POMx group and 33 11
days in the placebo group (two-sided t test for comparison
of means, P ¼ 0.280). With the exception of one subject for
whom a protocol deviation was approved for 75% compli-
Secondary outcomes
Urothithin A, a pomegranate metabolite, was detected
significantly more frequently in men in the POMx arm
(21/33 ¼ 64%) than in the placebo arm (12/35 ¼ 36%;
P ¼ 0.022). Moreover, when examined as a continuous
variable, Urolithin A levels were significantly higher in the
Results
Table 1. Baseline characteristics
Feature
Race
White
Black
Native American
Height, cm (mean SD)
Weight, kg (mean SD)
Age, y (mean SD)
Biopsy Gleason sum
6
7
8
9
ECOG
ECOG 0
PSA, ng/mL (Mean) SD
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POMx
Placebo
N (%)
N (%)
27 (81.8)
5 (15.2)
1 (3.0)
179.62 (8.733)
92.12 (17.070)
60.03 (7.935)
31 (86.1)
5 (13.9)
0 (0.0)
181.25 (12.001)
94.18 (15.836)
57.09 (6.254)
13 (39.4)
18 (54.5)
2 (6.1)
0 (0.0)
20 (55.6)
14 (38.9)
1 (2.8)
1 (2.8)
33 (100.0)
6.89 (3.884)
36 (100.0)
6.83 (4.274)
P
Test
c2
0.563
0.529
0.610
0.096
t test
t test
t test
c2
0.363
NA
0.878
No test conducted
Rank-sum test
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Table 2. Pathologic prostatectomy features
Feature
Surgical procedure
Open
Laparoscopic
Robotic
Unknown
T stage
pT1
pT2
pT3
Gleason at surgery
6
7
8
9
Surgical margins
Negative
Positive
Seminal vesicle involvement
No
Yes
N stage
pN0
pN1
pNx
POMx
Placebo
N (%)
N (%)
31 (93.9)
0 (0.0)
1 (3.0)
1 (3.0)
36 (100.0)
0 (0.0)
0 (0.0)
0 (0.0)
0 (0.0)
22 (60.7)
11 (33.3)
1 (2.8)
23 (63.9)
12 (33.3)
12 (36.4)
18 (54.5)
3 (9.1)
0 (0.0)
13 (36.1)
21 (58.3)
0 (0.0)
2 (5.6)
20 (60.6)
13 (39.4)
22 (61.1)
14 (38.9)
30 (90.9)
3 (9.1)
34 (94.4)
2 (5.6)
26 (78.8)
0 (0.0)
7 (21.2)
25 (69.4)
1 (2.8)
10 (27.8)
P
Test
c2
0.325
c2
0.626
c2
0.161
Fisher exact
1.000
c2
0.572
c2
0.491
more urolithin A associated with lower oxidative damage
as measured by 8-OHdG) in both benign (r ¼ 0.115, P ¼
0.369) and cancer tissue (r ¼ 0.299, P ¼ 0.017), though
this only reached statistical significance in the cancer tissue.
POMx group compared with placebo (1.12 vs. 0.49 ng/gm;
P ¼ 0.007). There were no differences between arms in PSA
before surgery (P ¼ 0.739) or in the ratio of baseline to
presurgery PSA (P ¼ 0.443). There were no between arm
differences in expression of pS6, NF-kB, or Ki67 within
prostate cancer tissue (Table 3).
Discussion
Though pomegranate extract and juice and POMx pills
have shown promise in preclinical and limited clinical
studies, there are limited data on the bioavailability and
distribution of orally consumed pomegranate and its in vivo
Exploratory outcomes
Among men from both arms combined, urolithin A levels
were inversely correlated with 8-OHdG expression (i.e.,
Table 3. Tissue analyses
1124
Therapy
N
Mean%
positive
SD
P
8-OHdG Normal cells
Placebo
POM
33
30
74.70
62.67
31.50
36.48
0.095
8-OHdG Tumor cells
Placebo
POM
33
30
33.52
25.90
38.45
33.76
0.372
pS6 Tumor cells
Placebo
POM
32
29
39.53
46.10
26.50
24.85
0.245
NF-kB Tumor cells
Placebo
POM
33
27
44.85
44.44
37.88
35.47
0.887
Ki67 Tumor cells
Placebo
POM
33
30
0.76
0.60
0.90
0.89
0.164
Cancer Prev Res; 6(10) October 2013
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POMx Before Radical Prostatectomy
cellular and molecular effects within prostate tissue (3–10).
In a randomized phase II trial of two daily tablets of POMx
versus placebo, we found that urolithin A, a pomegranate
metabolite, was significantly more likely to be present and
at higher levels in men assigned to POMx. This is the first
strong human evidence that orally administered pomegranate routinely reaches and accumulates in the prostate.
However, in this modest sized short-term study, POMx
treatment did not significantly lower 8-OHdG levels, a
measure of oxidative damage and our primary outcome.
Higher urolithin A levels, a key pomegranate metabolite,
were correlated with less 8-OHdG providing some evidence
to the hypothesis that pomegranates do in fact lower 8OHdG. As such, the current findings support further formal
hypothesis testing of POMx for reducing oxidative damage
as well as further animal testing to better understand the
multiple mechanisms through which POMx may alter prostate cancer biology.
Pomegranates have been touted to have numerous health
benefits (17). In regards to prostate cancer, several preclinical reports have shown that pomegranates, whether by
extract or concentrated juice, can slow prostate cancer
growth in vitro and in animal models (3–8). Unfortunately,
human studies of pomegranate for prostate cancer are
limited. To date, only three studies have been published
(9, 10, 18). In two of them, men with a rising PSA after
primary therapy were treated with pomegranate juice or
POMx (9, 10). Though neither study included a placebo
control, both studies found men who consumed pomegranates had longer PSADT values than before study enrollment, suggesting that pomegranate consumption may slow
human prostate growth. However, placebo-controlled trials
of men with rising PSA after primary therapy have also
shown that the majority of men treated with placebo have
longer on-study than prestudy PSADT (11), and thus
whether the longer PSADT truly reflected any anti-prostate
cancer activity of pomegranate is unclear. A randomized,
placebo-controlled study of pomegranate liquid extract
in men with rising PSA after primary therapy is nearing
completion.
Pomegranates have been shown to contain more than
100 different phytochemicals, including the bioactive family of ellagitannins (19). A number of studies have examined the oral bioavailability of pomegranate juice polyphenols (20, 21) determined by plasma bioavailability of
ellagic acid and urinary accumulation of urolithin A glucuronide, a urinary metabolite of ellagic acid. Pomegranate
ellagitannins are not absorbed intact into the blood stream
but are instead absorbed after being hydrolyzed to ellagic
acid in the intestine. Ellagitannins are also further metabolized into urolithins by gut flora, which are subsequently
conjugated in the liver and finally excreted in the urine. To
date, only one human study examined the effect of pomegranate consumption on prostate tissue (18). In this study,
19 men before surgery for either prostate cancer or benign
prostatic hyperplasia (BPH) were given pomegranate juice
for 3 days before surgery and were compared with 14
subjects given walnuts for 3 days before surgery and with
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30 untreated controls. Pomegranate juice was derived from
fresh pomegranates using a laboratory pilot press and
patients consumed 200 mL per day. Urolithin A was
detected in only 2 of the 19 men given pomegranate juice
compared with zero of the controls suggesting either limited
accumulation of pomegranate metabolites in the prostate or
lack of sensitivity in their detection. Though other tissue
analyses were limited, the authors did note no significant
differences in CDKN1a, Ki67, or c-Myc expression among
men treated with either pomegranate juice or walnuts versus
the untreated controls. Unfortunately, this study had
numerous limitations including a mixed group of men
undergoing surgery not just for prostate cancer but also for
BPH, small numbers (only 14 men treated with pomegranate), and the use of pomegranate for only a limited duration
of 3 days. As such, it is difficult to draw firm conclusions
from this one study. In contrast with this prior study, we
found that treatment for up to 4 weeks of a known pomegranate extract (POMx) resulted in significantly increased
urolithin A levels in the prostate. As such, we conclude that
treatment with POMx can result in detectable tissue levels of
a major pomegranate metabolite. However, it should be
noted that the overall levels of urolithin A were low consistent with the known poor uptake of ellagtannins and
ellagic acid in blood. Moreover, 12 subjects in the POMx
arm had undetectable levels. Whether this reflects different
pomegranate metabolism among different subjects, poor
compliance (though pill counting showed >75% compliance among all but one man), low overall POMx exposure,
or insufficient exposure time is unknown. However, these
findings suggest that future studies testing higher doses and
for longer duration of POMx may be warranted.
A large body of literature has linked inflammation and
the reactive oxygen species (ROS) generated secondary to
inflammation to prostate carcinogenesis (22). Inflammation in the microenvironment of the prostate cancer cell
may stimulate the multistep process of carcinogenesis by
upregulating the production of proinflammatory cytokines
and their signaling pathways. Evidence supports the concept that proliferative inflammatory atrophy of benign
prostate epithelium may be a precursor to prostatic intraepithelial neoplasia and prostate cancer (23). Inflammation
can result in persistent oxidative stress in cancer cells and the
ROS may lend cancer cells a survival advantage (24, 25).
Mild levels of oxidative stress stimulate cancer cell proliferation (24) and increase mutation rates through DNA
damage and/or epigenetic changes (26). Furthermore, low
levels of antioxidant enzymes and defective DNA repair of
oxidative DNA damage in malignant prostatic tissue relative
to benign prostate epithelium implicate oxidative DNA
damage in prostate carcinogenesis (23, 27).
Oxidative stress represents an imbalance between the
production and quenching of ROS, with accumulation of
intracellular free radicals that can damage all components
of the cell. Oxidative damage to the DNA base 20 -deoxyguanosine produces8-OHdG, a major product of DNA
oxidation. The concentration of 8-OHdG within a cell has
been proposed as a measurement of oxidant stress and
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Freedland et al.
oxidative DNA damage, and when it is incorporated into
DNA, 8-OHdG has shown a mutagenic potential, leading to
a point mutation via an A to T substitution. 8-OHdG levels
have been correlated with the incidence of several cancers
(28). We hypothesized that patients with prostate cancer
would exhibit a large amount of oxidized DNA adducts as a
result of GSTP1 gene inactivation and the chronic oxidant
stresses to which they are exposed. We also hypothesized
that the number of DNA adducts can be diminished by
treatment of patients with agents containing antioxidant
polyphenols such as POMx. Though the amount of oxidized
DNA adducts, such as 8-OHdG, present in the prostate of
patients with prostate cancer has not been established, a key
mechanism through which pomegranates are thought to
affect prostate cancer growth is via reducing oxidative damage (9, 13). Indeed, when prostate cancer cells are grown in
serum from men given pomegranate juice (Wonderful
variety), it results in less oxidative state and reduced oxidation of serum lipids versus cells treated with serum before
pomegranate intake (9). In the current study, we indeed
found that POMx treatment did lower 8-OHdG levels in
both benign (16% lower) and cancerous tissue (23%),
though this did not reach statistical significance in either
analysis. Moreover, higher urolithin A levels were correlated
with lower 8-OHdG levels further supporting the notion
that POMx may lower 8-OHdG. Unfortunately, how effectively POMx would alter 8-OHdG levels and a clinically
significant threshold in change in 8-OHdG levels were
unknown before the study, and thus we estimated our
power calculations and sample size upon prior studies of
different agents (15) and different tumor types (16) assuming that the extent of effect previously reported would be
similar compared with the proposed research. Thus,
although the current study failed to meet its primary endpoint, it does provide better effect estimates for powering a
larger study going forward. Moreover, it does suggest that
such an approach may be warranted.
We then examined other key prostate cancer biomarkers
including Ki67 (proliferation), pS6 (a marker of mTOR
activity), and NF-kB (a measure of inflammation). However, we found no effect on POMx on these relevant biomarkers. Of note, prior murine studies did show that
pomegranate can affect some of these markers (4, 6, 7). As
such, whether these negative data reflect insufficient dose or
duration of POMx therapy, or some other cause is
unknown. However, there are multiple putative mechanisms through which POMx may affect prostate cancer (2). As
such, further studies are needed to more comprehensively
investigate potential targets which are altered in response to
POMx therapy.
What we did confirm was the relative safety of shortterm POMx therapy. No patient had any adverse event at
grade 2 or higher. Consistent with the known side-effect
profile of POMx, we did have some mild gastrointestinal
effects (10). Thus, although continued efforts to determine the efficacy of POMx for prostate cancer are needed,
we can conclude based upon this study and prior clinical
trials of pomegranate juice and POMx in men with pros-
1126
Cancer Prev Res; 6(10) October 2013
tate cancer that it seems pomegranates are unlikely to be
harmful (9, 10).
This study is not without limitations. First, our primary
endpoint is an intermediate surrogate biomarker endpoint.
The clinical relevance of 8-OHdG levels is unclear. Thus, we
used 8-OHdG as a means to test whether POMx had "ontarget" effects which would support future placebo-controlled randomized studies aimed at more clinically relevant endpoints. Second, the number of men included was
modest limiting our statistical power to detect important
changes. Third, the duration of POMx therapy was short and
the dose was modest. As such, further studies are needed to
test whether longer duration or higher doses have greater
effects. This is particularly true in that we did find higher
urolithin A levels were correlated with lower 8-OHdG levels
suggesting higher doses may have greater effects within the
prostate. However, this analysis was limited by our inability
to separate the benign from the malignant tissue when
examining the urolithin A levels. Moreover, it is possible
that urolithin A levels were influenced by dietary sources
other than POMx tablets as indeed some men in the control
arm had detectable urolithin A levels. Future studies may
consider measuring urine urolithin A and other pomegranate metabolites as further controls assessing systemic
absorption. Finally, we only examined a small number of
secondary endpoints. It is hoped that future analyses of
these samples including full gene expression analyses
should yield valuable information about the effects of
POMx on the prostate.
Summary
A small randomized placebo-controlled phase II trial of
up to 4 weeks of dietary intervention with POMx before
radical prostatectomy did not significantly lower 8-OHdG
levels. However, the fact that urolithin A, an active pomegranate metabolite, was capable of absorption and accumulation in prostate tissues and higher urolithin A levels
correlated with lower 8-OHdG levels does provide some
evidence to support the underlying hypothesis that pomegranates may modulate 8-OHdG levels and suggests a role
for pomegranate juice in protection against oxidative DNA
damage. Further and larger studies with longer duration are
needed to formally test whether pomegranates can alter 8OHdG levels and the clinical relevance of this as well as
further animal testing to better understand the multiple
mechanisms through which POMx may alter prostate cancer biology.
Disclosure of Potential Conflicts of Interest
M.A. Carducci is a consultant/advisory board member of POM Wonderful. S. Kerkoutian is a consultant/advisory board member of POM Wonderful. D. Heber has commercial research grant from POM Wonderful. A.J.
Pantuck has commercial research grant from POM Wonderful. No potential
of conflicts were disclosed by the other authors.
Authors' Contributions
Conception and design: S.J. Freedland, M.A. Carducci, S. Kerkoutian, A.J.
Pantuck
Development of methodology: S.J. Freedland, N. Kroeger, S. Kerkoutian,
Y. Li, H. Fedor, D. Heber
Cancer Prevention Research
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Research.
Published OnlineFirst August 28, 2013; DOI: 10.1158/1940-6207.CAPR-12-0423
POMx Before Radical Prostatectomy
Acquisition of data (provided animals, acquired and managed patients,
provided facilities, etc.): S.J. Freedland, M.A. Carducci, A.W. Partin, J. Rao,
S. Kerkoutian, H. Wu, Y. Li, P. Creel, K. Mundy, R. Gurganus, H. Fedor,
Y. Zhang
Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): S.J. Freedland, M.A. Carducci, N. Kroeger,
J. Rao, S. Kerkoutian, A.J. Pantuck
Writing, review, and/or revision of the manuscript: S.J. Freedland, M.A.
Carducci, N. Kroeger, A.W. Partin, S. Kerkoutian, D. Heber, A.J. Pantuck
Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): S.J. Freedland, N. Kroeger, Y. Jin,
K. Mundy, H. Fedor, S. King, D. Heber, A.J. Pantuck
Study supervision: S.J. Freedland, M.A. Carducci, S. Kerkoutian, A.J.
Pantuck
Grant Support
This work was supported by POM Wonderful (to all authors), the Sense
Foundation (to all authors), DOD Clinical Consortium- W81XWH-09-10149 (to M.A. Carducci and S.J. Freedland), and the Johns Hopkins CORE
Grant-P30CA006973 (to M.A. Carducci).
The costs of publication of this article were defrayed in part by the
payment of page charges. This article must therefore be hereby marked
advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate
this fact.
Received October 15, 2012; revised August 5, 2013; accepted August 7,
2013; published OnlineFirst August 28, 2013.
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