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. Updated version Cited Articles E-mail alerts Reprints and Subscriptions Permissions Access the most recent version of this article at: doi:10.1158/1940-6207.CAPR-12-0423 This article cites by 27 articles, 9 of which you can access for free at: http://cancerpreventionresearch.aacrjournals.org/content/6/10/1120.full.html#ref-list-1 Sign up to receive free email-alerts related to this article or journal. To order reprints of this article or to subscribe to the journal, contact the AACR Publications Department at pubs@aacr.org. To request permission to re-use all or part of this article, contact the AACR Publications Department at permissions@aacr.org. Downloaded from cancerpreventionresearch.aacrjournals.org on May 2, 2014. © 2013 American Association for Cancer Research. 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 Cancer Prev Res; 6(10) October 2013 Downloaded from cancerpreventionresearch.aacrjournals.org on May 2, 2014. © 2013 American Association for Cancer Research. Published OnlineFirst August 28, 2013; DOI: 10.1158/1940-6207.CAPR-12-0423 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 www.aacrjournals.org 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 Cancer Prev Res; 6(10) October 2013 Downloaded from cancerpreventionresearch.aacrjournals.org on May 2, 2014. © 2013 American Association for Cancer Research. 1121 Published OnlineFirst August 28, 2013; DOI: 10.1158/1940-6207.CAPR-12-0423 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 Cancer Prevention Research Downloaded from cancerpreventionresearch.aacrjournals.org on May 2, 2014. © 2013 American Association for Cancer Research. Published OnlineFirst August 28, 2013; DOI: 10.1158/1940-6207.CAPR-12-0423 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 www.aacrjournals.org 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 Cancer Prev Res; 6(10) October 2013 Downloaded from cancerpreventionresearch.aacrjournals.org on May 2, 2014. © 2013 American Association for Cancer Research. 1123 Published OnlineFirst August 28, 2013; DOI: 10.1158/1940-6207.CAPR-12-0423 Freedland et al. 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 Cancer Prevention Research Downloaded from cancerpreventionresearch.aacrjournals.org on May 2, 2014. © 2013 American Association for Cancer Research. Published OnlineFirst August 28, 2013; DOI: 10.1158/1940-6207.CAPR-12-0423 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 www.aacrjournals.org 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 Cancer Prev Res; 6(10) October 2013 Downloaded from cancerpreventionresearch.aacrjournals.org on May 2, 2014. © 2013 American Association for Cancer Research. 1125 Published OnlineFirst August 28, 2013; DOI: 10.1158/1940-6207.CAPR-12-0423 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 Downloaded from cancerpreventionresearch.aacrjournals.org on May 2, 2014. © 2013 American Association for Cancer 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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