Cancer Therapy: Clinical

Phase II Study of Pomegranate Juice for Men with Rising

Prostate-Specific Antigen following Surgery or
Radiation for Prostate Cancer
Allan J. Pantuck,1 John T. Leppert,1 Nazy Zomorodian,1 William Aronson,1 Jenny Hong,1
R. James Barnard,3 Navindra Seeram,2 Harley Liker,2 Hejing Wang,4 Robert Elashoff,4
David Heber,2 Michael Aviram,5 Louis Ignarro,2 and Arie Belldegrun1

Abstract Purpose: Phytochemicals in plants may have cancer preventive benefits through antioxidation
and via gene-nutrient interactions. We sought to determine the effects of pomegranate juice

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(a major source of antioxidants) consumption on prostate-specific antigen (PSA) progression in
men with a rising PSA following primary therapy.
Experimental Design: A phase II, Simon two-stage clinical trial for men with rising PSA after
surgery or radiotherapy was conducted. Eligible patients had a detectable PSA >0.2 and <5 ng/mL
and Gleason score V7. Patients were treated with 8 ounces of pomegranate juice daily (Wonder-
ful variety, 570 mg total polyphenol gallic acid equivalents) until disease progression. Clinical end
points included safety and effect on serum PSA, serum-induced proliferation and apoptosis of
LNCaP cells, serum lipid peroxidation, and serum nitric oxide levels.
Results: The study was fully accrued after efficacy criteria were met. There were no serious
adverse events reported and the treatment was well tolerated. Mean PSA doubling time signifi-
cantly increased with treatment from a mean of 15 months at baseline to 54 months posttreat-
ment (P < 0.001). In vitro assays comparing pretreatment and posttreatment patient serum on
the growth of LNCaP showed a 12% decrease in cell proliferation and a 17% increase in apoptosis
(P = 0.0048 and 0.0004, respectively), a 23% increase in serum nitric oxide (P = 0.0085), and
significant (P < 0.02) reductions in oxidative state and sensitivity to oxidation of serum lipids after
versus before pomegranate juice consumption.
Conclusions: We report the first clinical trial of pomegranate juice in patients with prostate can-
cer. The statistically significant prolongation of PSA doubling time, coupled with corresponding
laboratory effects on prostate cancer in vitro cell proliferation and apoptosis as well as oxidative
stress, warrant further testing in a placebo-controlled study.

Adenocarcinoma of the prostate is currently the most common several years. Prostate cancer 5-year survival rates have
malignancy in men in the United States comprising 29% of all increased from 67% for the period of 1974 to 1976 to 92%
cancers. This year an estimated 232,090 men will be newly for the period of 1989 to 1995 (2). However, prostate cancer
diagnosed with prostate cancer (1). There has been a trend remains the second most common cause of cancer death in
toward improved survival in prostate cancer over the past men in the United States, accounting for 11% of all cancer
deaths. This year an estimated 30,350 men will die of prostate
cancer (1).
Primary management of prostate cancer for the majority of
Authors’Affiliations: Departments of 1Urology, 2Medicine, 3Physiologic Science, patients consists of either radical surgery or radiation therapy.
and 4Biomathematics, David Geffen School of Medicine, University of California at Although this is adequate for permanent disease control in
Los Angeles, Los Angeles, California and 5Technion Faculty of Medicine, Rambam many patients, a significant number of patients relapse and
Medical Center, Bat-Galim, Haifa, Israel
Received 10/21/05; revised 3/29/06; accepted 5/2/06.
ultimately develop metastatic disease. Radical prostatectomy is
Grant support: Lynda and Stewart Resnick Revocable Trust and grants currently the most commonly used therapy for curative intent
P50CA92131 and 1R01CA100938 (W. Aronson and R.J. Barnard). Lynda and (3). However, approximately one third of prostate cancer
Stewart Resnick own the POM Wonderful Co., which provided the juice for the patients with clinically confined cancer that are treated with
study.
radical prostatectomy will develop a biochemical recurrence
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 (4, 5). Pound et al. (6) reviewed 1,997 patients that underwent
with 18 U.S.C. Section 1734 solely to indicate this fact. radical prostatectomy for clinically localized prostate cancer
Requests for reprints: Allan J. Pantuck, Department of Urology, David Geffen and determined that 15% of patients had biochemical
School of Medicine, University of California at Los Angeles, 66-118 Center for recurrence. Thirty-four percent of patients with biochemical
Health Sciences, Box 951738, Los Angeles, CA 90095-1738. Phone: 310-206-
2436; Fax: 310-206-4082; E-mail: apantuck@ mednet.ucla.edu.
recurrence developed distant metastases with only 15 years of
F 2006 American Association for Cancer Research. total follow-up, median time to development of metastases was
doi:10.1158/1078-0432.CCR-05-2290 8 years from the time of initial prostate-specific antigen (PSA)

Clin Cancer Res 2006;12(13) July 1, 2006 4018 www.aacrjournals.org

 Pomegranate Juice and Prostate Cancer

elevation, and median time to death from the development of formation and growth in animals (17). Recently, there have
metastases was 5 years (6). been several reports on the antiproliferative, apoptotic,
There are limited treatment options for patients who have angiogenic, and inhibition of nuclear factor-nB (NF-nB)
undergone primary therapy with curative intent and who have activity and xenograft growth by pomegranate polyphenols
progressive elevation of their PSA without documented (18 – 22). Although these data on pomegranate polyphenols
evidence of metastatic disease. Early initiation of hormonal and NF-nB are quite preliminary and many mechanistic
ablation is associated with significant morbidity and effect on questions remain to be elucidated, it is consistent with a
quality of life, including fatigue, hot flashes, loss of libido, much larger body of literature that supports the concept that
decreased muscle mass, and osteoporosis with long-term use. plant polyphenols, including red wine resveratrol (23), green
Strategies to delay clinical prostate cancer progression and tea catechins (24), and soy isoflavones (25), are capable of
prolong the interval from treatment failure to hormonal decreasing proliferation and stimulating apoptosis via inhibi-
ablation would be of paramount importance. A combination tion of NF-nB activity. Finally, pomegranate phytochemicals
of epidemiologic and basic science evidence strongly suggests inhibited the in vitro proliferation of three prostate cancer cell
that diet and plant-derived phytochemicals may play an lines, LNCaP, PC3, and DU145, and showed in vivo
important role in prostate cancer prevention or treatment. inhibition of xenograft growth in athymic mice (20).
Multiple genetic and epigenetic factors have been implicated To study the possible therapeutic effects of pomegranate juice

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in the oncogenesis of prostate cancer. However, the molecular on prostate cancer, a single-center, open-label, phase II clinical
mechanism underlying the disease is not well understood. trial was done.
African American men have the highest rate of prostate cancer
in the world, whereas Japanese and Chinese native to their
countries who consume a low-fat and high-fiber diet with high Materials and Methods
consumption of phytochemicals that include soy products and
green tea have the lowest rate (7). Epidemiologic studies Clinical trial design. An open-label, single-arm clinical trial was
suggest that a reduced risk of cancer is associated with the done at the Clark Urologic Center, David Geffen School of Medicine,
consumption of a phytochemical-rich diet that includes fruits University of California at Los Angeles. A 2-year, single-center, phase II,
Simon two-stage clinical trial for men with rising PSA after surgery or
and vegetables [dietary aspects of both prostate cancer
radiotherapy was designed and done. Eligible patients had a detectable
prevention and treatment are well reviewed by Chan et al.
PSA >0.2 and <5 ng/mL that was documented as rising, enough
(8)]. Fresh and processed fruits and food products contain high pretreatment PSA time points to calculate a baseline PSA doubling time
levels of a diverse range of phytochemicals of which poly- (PSADT), no hormonal therapy before entering the study, no evidence
phenols, including hydrolyzable tannins (ellagitannins and of metastatic disease, and Gleason score V7.
gallotannins) and condensed tannins (proanthocyanidins), Serial PSA measurements before study entry determined a baseline
and anthocyanins and other flavonoids make up a large PSADT. Each patient had a minimum of three pretreatment PSA values
proportion (9). Several phytochemicals have been proposed measured over a minimum of 6 months before study entry. Patients
as potential chemoprevention agents based on animal and were treated with 8 ounces of pomegranate juice by mouth daily
laboratory evidence of antitumor effects. Suggested mecha- (Wonderful variety, equivalent to 570 mg total polyphenol gallic acid
equivalents daily) until meeting disease progression end points.
nisms of anticancer effects of polyphenols include the
Patients were followed in 3-month intervals for serum PSA, and blood
inhibition of cancer cell growth by interfering with growth and urine were collected for laboratory studies. Clinical end points
factor receptor signaling and cell cycle progression, promotion included safety, effect on serum PSA, effect on serum hormone levels
of cellular differentiation, modulation of phosphodiesterase/ (testosterone, estradiol, sex hormone-binding globulin, dehydroepian-
cyclooxygenase pathways, inhibition of kinases involved in cell drosterone, insulin-like growth factor, and androstenedione), and
signaling, and inhibition of inflammation (10 – 12). exploratory laboratory studies. These exploratory studies included
The pomegranate (Punica granatum L.) fruit has been used markers for treatment compliance (serum and urinary polyphenol/
for centuries in ancient cultures for its medicinal purposes ellagic acid levels), markers of serum antioxidant effect (serum nitrous
(13). Pomegranate fruits are widely consumed fresh and in oxide levels), and in vitro assays that measure the effect of patients’
beverage forms as juice and wines (14). Commercial serum on the LNCaP growth and apoptosis (26). For these laboratory
studies, each patient during treatment was compared with baseline.
pomegranate juice shows potent antioxidant (15) and
Samples were collected, processed, aliquoted, labeled with anonymized
antiatherosclerotic (16) properties attributed to its high study ID numbers, and batch stored at 80jC. For each assay,
content of polyphenols, including ellagic acid in its free and aliquoted samples were thawed and run simultaneously to ensure
bound forms (as ellagitannins and ellagic acid glycosides), standardization of conditions across samples. For each assay, pretreat-
gallotannins, and anthocyanins (cyanidin, delphinidin, and ment and posttreatment samples were randomly assayed by laboratory
pelargonidin glycosides) and other flavonoids (quercetin, technicians who were blinded to the corresponding clinical data. The
kaempferol, and luteolin glycosides; ref. 14). The most primary end point for this study was effect on PSA variables, such as
abundant of these polyphenols is punicalagin, an ellagitannin change in doubling time, and the secondary end points included safety
implicated as the bioactive constituent responsible for >50% and modulation of biomarkers. PSADT before treatment was compared
of the potent antioxidant activity of the juice (14). Punica- with the slope of this curve after treatment, because a significant change
in this slope may potentially correlate with delay in disease progression.
lagin is abundant in the fruit husk and, during processing, is
An objective response was defined as a decrease of z50% in highest
extracted into pomegranate juice in significant quantities measured serum PSA. Progressive disease was defined as either a >100%
reaching levels of >2 g/L juice (14). Ellagic acid and tannins increase in PSA (with a minimum value of 1.0 ng/mL) compared with
have been shown previously to exhibit in vitro and in vivo the best response observed (nadir) or any documentation of metastatic
anticarcinogenic properties, such as induction of cell cycle or recurrent disease. Patients with stable disease did not qualify as
arrest and apoptosis, as well as the inhibition of tumor objective response or progressive disease.

www.aacrjournals.org 4019 Clin Cancer Res 2006;12(13) July 1, 2006

Cancer Therapy: Clinical

The study was conducted according to the Declaration of Helsinki In vitro apoptosis assay. Experiments were carried out in 96-well
and its amendments. The study protocol was approved by the tissue culture plates at a density of 10  103 cells per well. Cells were
University of California at Los Angeles Medical Institutional Review prepared and plated for 24 hours as mentioned above. Using the Cell
Board and the Institutional Scientific Peer Review Committee of the Death Detection ELISA Plus (Roche Applied Science, Indianapolis, IN),
Jonsson Comprehensive Cancer Center. All patients gave written apoptosis was measured at the end of 2 days of treatment with 10%
informed consent before participation. fetal bovine serum or 10% experimental sera. This apoptosis assay is
The study sponsors played no role in the study design, collection, based on a quantitative sandwich enzyme immunoassay principle using
analysis, or interpretation of data or in the writing of this report. mouse monoclonal antibodies directed against DNA and histones and
Calculation of PSADT. The linear spline method with a knot at t = 0 allows the specific determination of mononucleosomes and oligonu-
(baseline) was used to estimate the PSADT before and after baseline cleosomes in the cytoplasm fraction of lysates. Background using
for each subject. The model is y = a + bt + ku(t), where y = log(PSA) incubation buffer and ABTS solution was subtracted from the
and u(t) = 0 if t < 0; otherwise, u(t) = t. PSADT is estimated: before absorbance measurements (405-490 nm). The results are expressed in
treatment, DT1 = log 2 / b; after baseline, DT2 = log 2 / (b + k). microunits of mononucleosomes and oligonucleosomes. Cell culture
Sample size and statistical considerations. The study was designed as and apoptosis assay were run in triplicates.
a two-stage optimal flexible design (27), phase II trial with the following Serum lipid peroxidation. Oxidative stress both in the basal state and
assumptions: the ineffectiveness cutoff was chosen equal to 5%; in an induced oxidative stress system [with the free radicals generator
the targeted desirable response rate cutoff equal to 20%. Hence, the 2,2-azobis(2-amidino-propane) dihydrochloride (AAPH)] was deter-
hypotheses of interest were H0: r0.05 against HA: r0.20, where r is the mined by measurement of serum lipid peroxidation in the patient’s

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response rate. The a error rate (probability of accepting an ineffective serum at baseline and after consumption of pomegranate juice.
treatment, a false-positive outcome) was set to 5%. The b error rate Serum samples were diluted 1:4 (v/v) with PBS and incubated for
(probability of rejecting an effective treatment, a false-negative outcome) 2 hours at 37jC without (basal state oxidative status) or with AAPH
was set to 10%. The study is designed to measure whether HA-H0 = (70 mmol/L; AAPH-induced oxidation). The extent of lipid peroxida-
0.15 with the following properties: if the true response rate is 5%, the tion was measured using 100 AL of the above solutions by the lipid
treatment is rejected with a very high probability (>1 a), whereas if the peroxide test, which analyzes lipid peroxide formation by their capacity
true response rate is 20%, the treatment is rejected with a very low to convert iodide to iodine, as measured spectrophotometrically at
probability (<b). In the first stage, 24 evaluable patients are treated. 365 nm (29).
If >1 patient achieved objective response or stable disease, additional The quantification of lipid peroxides, the major initial reaction
22 patients would be treated, up to a total of 46. At the end of the second products of lipid peroxidation, serves as a direct and valuable index of
stage, if <4 patients achieved objective response, the treatment would the oxidative status of biosystems. AAPH is a water-soluble azo-
be declared ineffective. Otherwise, the study is considered to have compound that is used extensively as a free radical generator in the
achieved its targeted desirable response rate (20%) and the treatment is characterization of antioxidants. Decomposition of AAPH produces
deemed promising for further testing in a phase III randomized study. molecular nitrogen and two carbon radicals. The carbon radicals may
Pomegranate juice processing. Pomegranate juice was provided by combine to produce stable products or react with molecular oxygen to
the Pom Wonderful Company (Los Angeles, CA). Pomegranates were give peroxyl radicals.
handpicked, washed, chilled to 4jC, and stored in tanks. The fruit was Evaluation of plasma nitric oxide. Concentrations of nitrite (NO2 )
then crushed, squeezed, and treated enzymatically with pectinase to and nitrate (NO3 ) were determined as an indirect measurement of
yield the juice and byproducts, which included the inner and outer nitric oxide (NO) production. Plasma nitrite and nitrate represent the
peels and the seeds. Flavonoids constitute 40% (anthocyanins, two stable oxidation products of NO in solution, and determination of
catechins, and phenols) of total polyphenols in pomegranate juice both anions represents an accurate quantitative measure of NO
(28). The pomegranate juice was filtered, pasteurized, concentrated, produced. A sensitive chemiluminescence detection procedure was
and stored at 20jC until use. used to measure plasma nitrite and nitrate. In short, plasma nitrite and
Treatment dose. Suggested clinical dosing of pomegranate juice was nitrate (NOx ) levels were measured by the classic Griess method. Total
based on its measured antioxidant effect in dose-response studies in nitrite was measured at 540 nm absorbance by reaction with Griess
healthy human males at the following content daily: 0 (baseline), 3, 6, reagent (sulfanilamide and naphthalene-ethylene diamine dihydro-
8, 12, and 15 ounces (equal to 90, 180, 240, 360, and 450 mL/d, chloride). Amounts of plasma nitrite were estimated by a standard
respectively). The results of these studies (not published) show that curve obtained from enzymatic conversion of NaNO3 to nitrite.
consumption of pomegranate juice by healthy male subjects exhibits Determination of ellagic acid and ellagic acid metabolites by liquid
gradual increased antioxidant capacities on using increasing dosages chromatography-electrospray ionization/mass spectrometry. Plasma
(0-15 ounces daily for 1 week). Based on these results, the from the centrifugation of collected whole blood and aliquoted urine
recommended clinical use of 6 to 8 ounces (180-240 mL) of [1.3 mL urine with 30 AL ascorbic acid/EDTA solution (20% ascorbic
pomegranate juice daily was suggested as the optimal dosage, with acid, 0.1% EDTA, NaH2PO4 0.04 mmol/L (pH 3.6-final pH 2))] were
significant clinical antioxidant effect and with no significant effect on collected at each study time point and stored immediately at 80jC.
serum triglycerides and glucose. The high-performance liquid chromatography system and conditions
Cell culture. Androgen-dependent LNCaP prostate tumor cells were for liquid chromatography-electrospray ionization/mass spectrometry
obtained from American Type Culture Collection (Manassas, VA). The are as described previously (30).
cells were grown in 75-cm2 flasks (Falcon Primaria, Bedford, MA) in
RPMI 1640 without phenol red, supplemented with 10% fetal bovine
Results
serum, 200 IU penicillin, 200 mg/mL streptomycin, and 4 nmol/L
L-glutamine (Omega Scientific, Inc., Tarzana, CA). The cultures were
The study was fully accrued to 48 participants over a period
maintained at 37jC and supplemented with 5% CO2 in a humidified
of 13 months in two stages after efficacy criteria were met.
incubator. Cells were passaged routinely at 80% confluence, and fresh
Two patients withdrew participation before their first evalu-
medium was replaced every third day. Cells used in experiments were
not passaged >10 times. ation, leaving 46 patients evaluable for treatment response. Of
In vitro proliferation assay. The effect on the in vitro proliferation of the patients enrolled, 68% were originally treated by radical
LNCaP by the exchange of culture medium supplemented with 10% prostatectomy, 10% by external beam radiotherapy, 10% by
fetal bovine serum with medium supplemented with subject serum was brachytherapy, 7% by surgery and radiation, and 5% by
determined as described previously (26). cryotherapy. The original Gleason score were read as

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 Pomegranate Juice and Prostate Cancer

intermediate (5 – 7) in 94% of patients, whereas 6% had Gleason rank sum test, P = 0.0001). Due to the large number of patients
4 cancers. All patients were clinical or pathologic N0 and M0, that continued to have stable disease at 18 months of
and 63% were clinically or pathologically staged with treatment, the protocol was amended to permit patients to
organ-confined disease, whereas 37% had locally advanced continue treatment until meeting disease progression criteria.
cancers extending into the periprostatic or seminal vesicle tissues. At 33 months, 3 additional patients were able to be included
At study entry, median PSA for the cohort was 1.05 ng/mL for analysis, as their PSA values had increased to the point that
(average, 2.23 F 2.58 ng/mL). they developed positive PSA slopes, allowing their PSADT to be
In stage I, 24 patients were treated of whom 2 patients were estimated. At 33 months, the estimate of the average PSADT
objective responders, with PSA declines of 85% and 50%. In the before intervention was 15.6 F 10.8 months (median, 11.5
first responder, the PSA rose to a maximal value at 6 months on months), whereas the average posttreatment PSADT was 54.7 F
study, followed by the 85% decrease in PSA level, which did 102 months (median, 28.7 months; P < 0.001). During the
not return to its peak until 21 months. The second responder study, 83% of patients achieved an improvement in PSADT
continues to maintain a 57% reduction in peak PSA at (i.e., either prestudy PSADT > 0 and poststudy PSADT < 0 or
33 months on study. In addition to these 2 subjects, 6 of the poststudy PSADT > prestudy PSADT) after intervention (signed
first 24 patients progressed by PSA criteria. Of the 46 patients rank sum test; P < 0.0001). Baseline T stage showed some
analyzed from both stages, 16 (35%) achieved a decrease in correlation with the change of PSADT, but this did not reach

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PSA during treatment (median, 18%; average, 27%; range, statistical significance. There were no serious adverse events
5-85%). Of these 46 patients, a total of 4 met objective reported and the treatment was well tolerated, and no patients
response criteria and achieved a PSA decline >50%, meeting developed metastatic disease on study.
efficacy criteria to justify future clinical testing. The slope of The effects of pomegranate juice treatment on in vitro cancer
their mean log PSA decreased 35%, from 0.066 F 0.007 (mean cell growth from baseline and posttreatment patient serum was
k F SE) at baseline down to 0.043 F 0.007 (mean k F SE) on compared using a cell culture assay system designed to measure
treatment (P < 0.001; Fig. 1). the effects of medium supplemented with individual serum on
Comparisons between baseline and posttreatment PSADT the growth of human prostate cancer cells. This cell culture
were calculated at multiple time points. At 15 months, there assay, developed in our laboratory (W.A. and R.J.B.), tests in an
were 28 patients with sufficient data points for meaningful exploratory manner whether pomegranate juice consumption
analysis. At 15 months, the average pretreatment baseline results in serum changes that reduce the growth or increase
PSADT was estimated to be 14.3 F 10.8 months, whereas the apoptosis in prostate cancer cell lines in vitro. To perform this
posttreatment PSADT was 25.5 F 33.5 months (P = 0.048). At assay, fetal bovine serum in LNCaP tissue culture medium was
24 months, the average PSADT for 39 patients before replaced with subject serum as described previously (26).
intervention was 15.0 F 11.1 months (median, 11.5 months). Compared with baseline, at 9 months, there was a 12%
At 24 months, 7 subjects had negative slopes (PSA was reduction (Fig. 2) in the growth of LNCaP (signed rank sum
decreasing); therefore, no PSADT could be estimated in these test; P = 0.0048), with 84% of patients showing a month 9
patients who were therefore excluded from analysis at this time value that was less than their respective baseline (mean
point. Note that by excluding patients with a declining PSA decrease in proliferation posttreatment, 0.024 F 0.075).
from the PSADT calculation the actual PSADT will be under- Interestingly, the postintervention serum in 6 subjects, of which
estimated. In the remaining evaluable patients, the PSADT was 3 were classified as having progressive disease, increased the
37.0 F 53.0 months (median, 19.9 months). The 22-month proliferation of LNCaP cells. The results of these bioassays
prolongation in PSADT reached statistical significance (signed theoretically represent the total patient growth factor milieu
comprising all proapoptotic/antiapoptotic factors in the sub-
jects fasting serum, with multiple factors in the serum not
related to pomegranate intake also potentially affecting their
results. These changes noted in cellular proliferation were
associated with a corresponding 17.5% increase in apoptosis at
9 months (Fig. 3) compared with baseline (signed rank sum
test; P = 0.0004), with 75% of patients tested showing an
increase in apoptosis (mean increase in apoptosis posttreat-
ment, 0.010 F 0.018). Baseline apoptosis and proliferation
were not significantly correlated with baseline PSA level.
The in vitro serum antioxidant effect of pomegranate juice
consumption was determined by evaluating the basal serum
oxidative state and the sensitivity to AAPH-induced oxidation
of the patients’ serum at baseline and after 9 months of
pomegranate juice consumption using the lipid peroxides
method. Compared with baseline, patients’ serum showed a
significant 40% reduction in the basal oxidative state and a
Fig. 1. Each individual thin line represents the log PSA by time for one subject,
significant 15% reduction in the resistance of their serum
pretreatment and posttreatment (month 0 = baseline treatment), with the samples to AAPH-induced lipid peroxidation after pomegranate
average slope of the entire cohort plotted in thick black line. The PSA values tend juice consumption (P < 0.02 for both tests; Fig. 4).
to increase, but the increase rate (slope) decreased. The slope of the mean log PSA
of the entire cohort decreased 35%, from 0.066 F 0.007 (mean k F SE) at Compared with baseline, there was a 23% increase in serum
baseline down to 0.043 (mean k F SE) on treatment (P < 0.001). NO metabolites measured in patients serum at 9 months

www.aacrjournals.org 4021 Clin Cancer Res 2006;12(13) July 1, 2006

Cancer Therapy: Clinical

Fig. 2. Percent change in the growth of human prostate cancer cells (LNCaP)
using culture medium supplemented with individual serum, comparing proliferation
observed using posttreatment versus baseline patient serum. Compared with
baseline, at 9 months, there was an average 12% reduction in the growth of LNCaP
(signed rank sum test, P = 0.0048).

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(mean of difference, 8.56 Amol/L; P = 0.0085), with two thirds
of patients assayed having an increase compared with baseline
(Fig. 5). There was a significant negative correlation between
baseline PSA and baseline nitrite level (Spearman correlation
coefficient = 0.40; P = 0.0281).
Pomegranate polyphenols (i.e., ellagic acid) were detected in
the urine of all participants by liquid chromatography-mass
spectrometry (Fig. 6).
There was no significant difference in pretreatment and
posttreatment patient hormone levels (testosterone, estradiol,
sex hormone-binding globulin, dehydroepiandrosterone, insu-
lin-like growth factor, and androstenedione) measured (data
not shown).
Fig. 4. Oxidative state and sensitivity to oxidation of serum from prostate cancer
patients before and after pomegranate juice consumption. A, basal oxidative state
was analyzed in the patients’ serum samples by the lipid peroxide method before
Discussion and after pomegranate juice (PJ) consumption. B, resistance of the serum samples
to the free radical generator AAPH-induced lipid peroxide formation was analyzed
In this study of 46 men with recurrent prostate cancer, by the lipid peroxides method. Columns, mean; bars, SD. *, P < 0.02.
treatment with pomegranate juice was associated with statisti-
cally significant prolongation of PSADT (albeit it, with
inverse relationship between diet and these prostate cancer
relatively few objective PSA responses), durable prolongation
end points has been epidemiologic in nature, primarily
of disease stabilization, and significant effects on exploratory
observational cohort and case-control studies (8). The largest
laboratory assays, such as the patients’ serum antioxidant
prospective study of this type, the European Prospective
status, and on prostate cancer in vitro cell growth and apoptosis.
Investigation into Cancer and Nutrition, is ongoing. European
The pattern of achieving a slowing of PSA progression without
Prospective Investigation into Cancer and Nutrition is the
significant PSA declines is consistent with a cytostatic rather
largest study of the relationship between diet, nutritional
than a cytotoxic treatment effect.
status, environmental factors, and incidence of cancer, having
There has been a great deal of interest regarding the potential
recruited >500,000 participants in 10 European countries
role of diet in altering the incidence, progression, or mortality
beginning in 1992. Preliminary, early results from the European
of prostate cancer. Much of the research that has shown an
Prospective Investigation into Cancer and Nutrition study
have not shown a link between the self-reported amount of

Fig. 3. Percent change in apoptosis of human prostate cancer cells (LNCaP)

using culture medium supplemented with individual serum, comparing cell death
observed using posttreatment versus baseline patient serum. Compared with Fig. 5. Percent change in circulating serum NO metabolites. Compared with
baseline, at 9 months, there was a 17.5% increase in apoptosis at 9 months baseline, there was a 23% increase in serum NO metabolites measured in patients
compared with baseline (signed rank sum test, P = 0.0004). serum at 9 months (P = 0.0085).

Clin Cancer Res 2006;12(13) July 1, 2006 4022 www.aacrjournals.org

 Pomegranate Juice and Prostate Cancer

Fig. 6. Liquid chromatography-electrospray

ionization/mass spectrometry spectra of
posttreatment patient urine showing
extracted ion chromatogram of ellagic acid,
M-H m/z 301. Spectra were obtained by
electrospray ionization in negative mode
acquiring ions between 120 and 1,500

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a.m.u.

fruits and vegetables that men eat and their risk of developing speculate on the potential mechanisms of action of pome-
prostate cancer at 4.8 years (31). Several large, prospective, granate polyphenols on the growth dynamics observed in the
randomized, placebo-controlled experimental studies to study in vivo prostate cancer assays. The lack of any observed effect
dietary prevention of prostate cancer are ongoing, including on serum androgen levels suggests that the effect is not
the Selenium and Vitamin E Chemoprevention Trial sponsored primarily hormonal in nature. Further, there is no reason to
by the NIH, and a study of selenium in men with high- believe that polyphenols artifactually affect circulating serum
grade prostatic intraepithelial neoplasia organized by the PSA measurement. This is supported by a separate pharmaco-
Southwest Oncology Group. Although much focus has been kinetic study (data not shown), in which there was no
on identifying primary chemopreventive agents that might relationship between circulating ellagic acid and PSA when
delay or prevent cancer from developing de novo, there is no both were serially and simultaneously measured after pome-
a priori reason to assume that such an agent would be as granate juice consumption. Chief, however, among the
effective in the treatment of established cancer or vice versa. potentially explanatory hypotheses is the role of inflammation
Unfortunately, as of yet, there have been no comparably in cancer and the antioxidant and anti-inflammatory effects of
sized clinical intervention studies of dietary treatments for pomegranate polyphenols. Chronic inflammation has been
established prostate cancer, although several small, PSA-based linked to the incidence of many cancers, including that of the
phase II studies have been reported. These studies include prostate (37). Studies investigating samples of human tissues
foods, such as soy protein (32, 33), mushroom extract (34), have shown that epithelial cell proliferation is increased by
and multi-ingredient dietary antioxidant nutritional supple- inflammation (38). An increased risk of cancer is associated
ments (35, 36). Although with few exceptions (33, 36) PSA with inflammatory mechanisms, as f15% of all cancers can be
kinetics were unaffected by the dietary treatment studied, related to chronic inflammation (38). Epidemiologic studies
these studies have tended to feature small cohorts of patients, have found an increased risk for prostate cancer in men who
treated for short periods of time, and without placebo control have a prior history of sexually transmitted disease or prostatitis
comparison. (39). Inflammation in the microenvironment of the prostate
In the current study, in addition to the significant prolon- cancer cell may stimulate the multistep process of carcinogen-
gation of PSADT, pomegranate juice intake was associated esis by up-regulating the production of proinflammatory
with antiproliferative and proapoptotic effects in our explor- cytokines and their signaling pathways. In fact, proliferative
atory bioassays. It should be recognized that the clinical inflammatory atrophy has recently been proposed as a
relevance of these unvalidated assays is as yet unclear. However, precursor to prostatic intraepithelial neoplasia and prostate
although the specific factors mediating these effects will be a cancer (40).
subject of future studies in a prospective randomized trial Inflammation can result in persistent oxidative stress in
comparing pomegranate juice with placebo, it is interesting to cancer cells and the reactive oxygen species may lend cancer

www.aacrjournals.org 4023 Clin Cancer Res 2006;12(13) July 1, 2006

Cancer Therapy: Clinical

cells a survival advantage (41 – 43). Mild levels of oxidative apoptosis and showed in vivo inhibition of xenograft growth
stress stimulate cancer cell proliferation (42) and increase in athymic mice (20). Also in PC3, Malik et al. (52) showed
mutation rates through DNA damage and/or epigenetic inhibition of cell growth by 3-(4,5-dimethylthiazol-2-yl)-2,5-
changes (44). De Marzo et al. (40) have shown the loss of diphenyltetrazolium bromide assay and induction of apoptosis
glutathione S-transferase P1 as an early event in prostate as determined by DNA fragmentation, induction of Bax and
tumors that sets the stage for stimulation of growth by oxygen Bak, and down-regulation of Bcl-XL and Bcl-2. Likewise, we
radicals. Oxidative stress has also been shown to increase cancer have presented preclinical in vitro studies6 that have shown
cell proliferation by increasing the sensitivity of growth factor 59% to 75% growth inhibition, delayed progression into S
receptors and by altering transcription factor activity. Inflam- phase, and generation of low levels of apoptosis in the PC3
matory cells, such as macrophages and mast cells, release prostate cancer cell line, whereas s.c. and orthotopic in vivo
angiogenic factors and cytokines like tumor necrosis factor-a, studies of LAPC-9 tumors in severe combined immunodeficient
interleukin-1, and vascular endothelial growth factor, which mice have shown 52% growth inhibition tumors 70%
signal cell growth and proliferation. Additionally, cytokines reduction in PSA.7
regulate signaling pathways that control proliferation, apopto- Although a plethora of assays are available to measure
sis, differentiation, and metastasis. oxidative stress and antioxidant status (53, 54), currently no
Oxidative stress is thought to be a factor associated with standard methods are available for estimating oxidative stress,

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many human diseases either as a cause or as an effect. Diverse making the choice of assay somewhat arbitrary. To date, the
diseases, including cancer, cardiovascular disease, and diabetes overwhelming majority of these assays have not been subjected
mellitus, are associated with oxidative damage mediated to rigorous validation checks, and none can currently be
presumably via reactive oxygen and nitrogen species (reactive classified as a true clinical efficacy measure or as a validated
oxygen and nitrogen species, respectively). The main cellular surrogate end point. This is true even for perhaps the most
components susceptible to damage by free radicals are lipids commonly used and popular assays of oxidative damage,
(peroxidation of fatty acids in membranes), proteins (denatur- 8-oxo-7,8-dihydroguanine, which measures the extent of DNA
ation), and nucleic acids. The role of antioxidants derived from base oxidation. Although the European Standards Committee
the diet in protection against oxidative stress and the on Oxidative DNA Damage was set up to resolve the problems
development or progression of cancer remains a topic of associated with the measurement of background levels of
significant controversy. The antioxidative properties of plant oxidative DNA damage, large sources of uncertainty still exist in
polyphenols are thought to arise from their reactivity as estimating concentrations of 8-oxo-7,8-dihydroguanine.
hydrogen or electron donors, from their ability to stabilize In the current study, two assays were used to estimate
unpaired electrons, and from their ability to terminate Fenton oxidative stress, one directly through a conventional assay
reactions (45). Although many dietary antioxidants have been (lipid peroxidation) and the other indirectly through an
shown to function as antioxidants in vitro, the ability of estimate of NO, which has well-defined anti-inflammatory
these substances to decrease oxidative damage to biomolecules properties (55). It has been shown previously that intervention
in vivo in humans has been less frequently studied, and long- with antioxidants can increase endothelial NOS expression
term intervention studies using manipulation of dietary both in cultured endothelial cells and in hypercholesterolemic
antioxidants have been done even less frequently mainly with mice (56). More recently, it has been reported that polyphenol
disappointing results (46, 47). flavonoids contained in pomegranate juice are capable of
Potent antioxidant and prostaglandin-inhibitory activities eliciting similar effects, enhancing the biological actions of
have been suggested previously for polyphenols extracted from NO by virtue of their capacity to stabilize NO. By protecting
pomegranate juice and seed oil, although the literature on this against the oxidative destruction of NO by reactive oxygen
topic remains relatively small (48). Likewise, however, these species and other radicals, pomegranate juice has produced
polyphenols have been shown to promote apoptosis, to inhibit higher and more prolonged cellular NO concentrations and
proliferation and invasion, and to inhibit 7,12-dimethylben- biological actions (57). The role of NO as a potential
z(a)anthracene – initiated carcinogenesis in vitro and in vivo chemopreventive agent remains controversial, with both
models of human and murine breast cancer (22). In skin cancer inhibitory and promoting effects on neoplasia being reported
models, pomegranate seed oil significant decreased tumor secondary to the NO/inducible NOS system (58). In studies
incidence and multiplicity in CD1 mice and was associated in vitro, NO-donating aspirin, consisting of aspirin plus an
with an inhibition of 12-O-tetradecanoylphorbol-13-acetate – -ONO(2) moiety linked via a molecular spacer, has been
induced ornithine decarboxylase activity (49) as well as shown to inhibit the growth of colon, pancreatic, lung,
modulation of mitogen-activated protein kinase and NF-nB skin, leukemia, breast, and prostate cancer cells (59), owing
pathways (18). The suppression of NF-nB is notable and has both to inhibition of cellular proliferation by blocking the G1-S
been noted that pomegranate can suppress NF-nB activation in cell cycle transition and to induction of apoptosis at least
other models, such as vascular endothelial cells (50). Activation partially through inhibition of NF-nB. In this current study,
of the NF-nB transcription factor results in up-regulation of there was a significant negative correlation between baseline
antiapoptotic genes, and NF-nB is thought to be a key factor PSA and baseline serum nitrite level (Spearman correlation
related involved in the control of cell proliferation, inhibition coefficient = 0.40; P = 0.0281).
of apoptosis, and oncogenesis in many cancer, including
prostate cancer (51). Pomegranate phytochemicals have been
shown to inhibit the in vitro proliferation of three prostate 6
Unpublished data.
cancer cell lines, LNCaP, PC3, and DU145, mediated by 7
A.J. Pantuck and A.S. Belldegrun. Prostate Cancer Foundation Scientific Retreat,
changes in both cell cycle distribution and induction of LakeTahoe, NV, 2004.

Clin Cancer Res 2006;12(13) July 1, 2006 4024 www.aacrjournals.org

 Pomegranate Juice and Prostate Cancer

From a practical standpoint, the prevention of disease and the need for confirmatory prospective studies using a
progression to a metastatic state, where the risk of cancer blinded control arm.
related mortality is increased, is also a desirable outcome. This
objective can be achieved by eliminating the disease but also Conclusions
by slowing the growth of the tumor. It remains controversial
whether modulation of PSA levels represents an equally valid We report the first clinical trial of pomegranate juice in
clinical end point. Although the Food and Drug Administration patients with recurrent prostate cancer. This study shows
has not yet accepted a PSA end point to support drug approval, statistically significant effects on PSADT coupled with
evaluation of additional data and further discussions of PSA corresponding effects on prostate cancer in vitro cell growth
end points are planned in future workshops and Oncologic and apoptosis. These proposed benefits, however, are in assays
Drugs Advisory Committee meetings. PSADT, however, is that are as yet unvalidated, and further research is needed to
increasingly being seen by some as an important surrogate prove the validity of these tests and to determine whether
biomarker for prostate cancer mortality. Recently, it was improvements in such biomarkers (including PSADT) are likely
shown that a posttreatment PSADT of <3 months seems to to serve as surrogates for clinical benefit. The present results,
be a surrogate end point for prostate cancer-specific mortality therefore, are being further tested in a randomized, double-
(60). Freedland et al. recently published the outcomes of blind, three-arm, placebo-controlled study, in which the ability

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379 prostate cancer patients who developed an increasing PSA of two pomegranate juice doses to produce a predefined
level after radical prostatectomy, finding that in this setting alteration in PSA kinetics is compared with the change
PSADT is the clinical factor most consistently correlated with observed in a control group. This future study, which began
death from prostate cancer (61). Because men with a greater in April 2006, addresses several limitations of the current study,
PSADT can expect a longer survival, implementation of with the inclusion of two treatment arms in a dose-response
strategies that could prolong PSADT, thereby delaying the time design as well as the use of a placebo control. Furthermore, the
to treatment with hormone therapy and to death, would of planned study will be used to prospectively evaluate the
great benefit to patients. Previously, several small single-arm performance of the exploratory biomarkers used in the current
phase II studies of agents in prostate cancer have shown protocol and to permit further study of potential serum factors
stabilization of PSA variables (62 – 64), but at least one such mediating the antiproliferative and proapoptotic effects ob-
study failed to show a statistically significant effect on the served in the present study. Finally, should such a study be
PSADT when compared in a placebo-controlled study (65). positive, a strong rationale would exist for research on other
Interestingly, in the randomized study of the peroxisome plant polyphenols (red wine resveratrol, etc.) that might
proliferator-activated receptor-g inhibitor rosiglitazone in men mediate similar effects.
with prostate carcinoma and a rising serum PSA level, 38% of
men in the placebo arm experienced a favorable outcome,
defined as a posttreatment PSADT >150% of the baseline
Acknowledgments
PSADT. Although this incidence of PSADT prolongation was We thank Jeffrey Gornbein (Department of Biomathematics, University of Cali-
smaller than that seen in this study, these studies highlight the fornia at Los Angeles) for additional statistical assistance and Randy Caliliw for
potential limitations of PSA variables in monitoring patients management of all tissue samples and coordination of laboratory assays.

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