Lycopene inhibits DNA synthesis in primary prostate ... - Nature

Prostate Cancer and Prostatic Diseases (2006) 9, 407–413 & 2006 Nature Publishing Group All rights reserved 1365-7852/06 $30.00 www.nature.com/pcan

ORIGINAL ARTICLE

Lycopene inhibits DNA synthesis in primary prostate epithelial cells in vitro and its administration is associated with a reduced prostate-specific antigen velocity in a phase II clinical study NJ Barber1, X Zhang2,3, G Zhu3, R Pramanik2, JA Barber4, FL Martin5, JDH Morris2 and GH Muir3 1

Department of Urology, Frimley Park Hospital, Surrey, UK; 2The Rayne Institute, King’s College London, London, UK; Department of Urology, King’s College Hospital, London, UK; 4Department of Medical Statistics, University College Hospital, London, UK and 5Department of Biological Sciences, IENS, Lancaster University, Lancaster, UK

3

Interest in lycopene has focused primarily on its use in the chemoprevention of prostate cancer (CaP); there are few clinical trials involving men with established disease. In addition, most data examining its mechanism of action have been obtained from experiments using immortal cell lines. We report the inhibitory effect(s) of lycopene in primary prostate epithelial cell (PEC) cultures, and the results of a pilot phase II clinical study investigating whole-tomato lycopene supplementation on the behavior of established CaP, demonstrating a significant and maintained effect on prostatespecific antigen velocity over 1 year. These data reinforce the justification for a large, randomized, placebo-controlled study. Prostate Cancer and Prostatic Diseases (2006) 9, 407–413. doi:10.1038/sj.pcan.4500895; published online 19 September 2006

Keywords: prostate cancer; lycopene; diet

Introduction Issues relating to male health, including prostate disease, continue to become more prominent in the public eye. Prostate cancer (CaP) is being diagnosed more frequently following the advent of plasma prostate-specific antigen (PSA) testing. Nevertheless, adenocarcinoma of the prostate remains the second biggest cancer killer of men.1 Owing to greater public access to information, the male population is becoming more aware of the risk of developing this disease. In recent years, there has been increasing interest in the possible role of dietary factors in the development and progression of cancers, including CaP, based on mounting circumstantial epidemiological and scientific data. As a consequence, it is claimed that a number of dietary supplements prevent CaP or reduce its rate of progression. Among these dietary supplements are included selenium, vitamin E, genestein and lycopene. Indeed, significant numbers of men diagnosed with CaP admit to having tried or taken health food supplements with a view to treating their cancer.2 Lycopene, a member of a group of natural pigments known as the carotenoids, is a powerful dietary antioxidant. It can be synthesized by plants or microorganisms and is widely found in the environment, giving red or yellow color to a number of plant species. Correspondence: Dr NJ Barber, Department of Urology, Frimley Park Hospital, Portsmouth Road, Camberley, Surrey GU16 5UJ, UK. E-mail. [email protected] Received 11 January 2006; revised 25 April 2006; accepted 5 May 2006; published online 19 September 2006

For humans, lycopene is found in a relatively narrow range of foods and the principal dietary source for most people is tomatoes. Dietary lycopene has been shown to accumulate in a number of specific organs in the human body, including the liver, prostate and adrenal glands. A number of epidemiological studies have suggested an inverse relationship between dietary lycopene intake and the risk of developing CaP. This has been supported by in vitro experiments using PC3, LnCaP and DU145 immortalized CaP cell lines, which demonstrate that lycopene at physiological concentrations inhibits cell proliferation.3 Although clinical CaP has a fairly typical behavior, most commercially available CaP cell lines (the exception being LnCaP cells that do secrete PSA) do not show a recognizable ‘prostatic’ phenotype. Such cell lines exhibit many abnormal growth characteristics that make them flawed surrogates to predict the behavior of prostate epithelial cells (PECs) in vivo and the use of these continuous cell lines of transformed phenotype should be treated with caution. In order to improve the reliability and relevance of cell culture experiments, we employed in this study primary PECs isolated from tissues (n ¼ 6) obtained at the time of prostatic surgery and examined the effect of lycopene on DNA synthesis as measured by the incorporation of 5-bromo-2-deoxyuridine (BrdU). As a result of these preliminary investigations, we proceeded to a small-scale study on patients (n ¼ 41) previously diagnosed with CaP to investigate if supplementary dietary lycopene would retard the rate of progression of the disease as judged by changes in serum PSA levels. Published clinical trials of this type are rare.

DNA synthesis in primary prostate epithelial cells in vitro NJ Barber et al

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Materials and methods Cell culture experiments – materials With approval from the local ethical committee (LREC no. 01-242 Research Ethics Committee, King’s College Hospital), patients (n ¼ 6) undergoing prostatic surgery (radical prostatectomy for organ-confined CaP) were approached for recruitment. After gaining consent, histologically benign prostate tissue was obtained at the time of surgery without complication. Dulbecco’s modified Eagle’s medium (DMEM), Roswell Park Memorial Institute (RPMI) 1640 medium, mitomycin c (MMC), Type 1A collagenase, lycopene, tetrahydrofuran (THF) and BrdU were obtained from Sigma-Aldrich (St Louis, MO, USA). NIH-3T3 fibroblasts were purchased from American Type Culture Collection. PEC basal medium (PrEBM) and 2.5% trypsin/versene solution were obtained from Cambrex Company (East Rutherford, NJ, USA). Fetal calf serum (FCS) and phosphate-buffered saline (PBS) were purchased from Invitrogen (Carlsbad, CA, USA). Monoclonal anti-BrdU antibody and propidium iodide (PI) were obtained from Amersham-Pharmacia (Buckinghamshire, UK) and goat anti-mouse Ig(H þ L)-FITC antibody obtained from Southern Biotechnology (Birmingham, AL, USA). Lycopene powder was stored at 701C. A 1 mM lycopene stock solution in THF was prepared under dark room conditions before use. Preparation of primary PECs In six-well plates containing 22-mm coverslips precoated with collagen (10 mg/ml in 1 mM glacial acetic acid), NIH-3T3 cells were grown in DMEM/10% FCS and treated overnight with 40 mg/ml MMC. Immediately following surgical resection, prostate tissue was transferred into RPMI 1640 medium supplemented with 20 mM 2-[4-(2-hydroxyethyl)-1-piperazinyl]ethanesulfonic acid (pH 7.4), 5% FCS, penicillin (10 U/ml), streptomycin (10 mg/ml) and amphotericin B (2.5 mg/ml). After transferal to the laboratory, the tissue was washed with sterile PBS and cut into o1-mm3 pieces before three further washes in PBS. Tissues were then digested with Type 1A collagenase (5 mg/g of tissue in 10 ml RPMI 1640) at 371C with gentle agitation for 18–20 h. Collagenase-treated tissues were centrifuged and washed in PBS and further digested with 0.25% trypsin/versene solution (20 ml) at 371C with gentle agitation for 20–30 min. Cell digests were centrifuged and re-suspended in RPMI 1640/10% FCS medium. Cells were counted, re-suspended in PrEBM and seeded at 4 104 cells per coverslip together with 105 MMC-treated NIH-3T3 cells (lethally inactivated) in 3 ml PrEBM; PECs were grown at 371C in 5% CO2. After 3 days, detached 3T3 feeder cells were removed by aspiration and PECs re-fed with fresh PrEBM to be used 7 days later. To confirm that the cultures consisted of PECs, conditioned medium was taken and analyzed for the presence of secreted PSA. Assay for DNA synthesis DNA synthesis was examined by determining the percentage of cells that incorporated BrdU into DNA. PECs were placed in fresh PrEBM and treated for 48 h with lycopene, as indicated, before the addition of 10 mM

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BrdU for an additional 4 h. Cultures were fixed for 20 min at room temperature with 3.7% formaldehyde in PBS and cells permeabilized with 0.2% Triton X-100 (5 min), washed with PBS, treated with 2 M HCl (10 min), washed with PBS, treated with 100 mM Tris (pH 7.8, 5 min) and then washed again with PBS. For immunostaining, cells were incubated with mouse monoclonal anti-BrdU diluted 1:25 in 20% goat serum/PBS for 1 h at room temperature and then washed with PBS. Cells were stained with goat anti-mouse antibody coupled to fluorescein-5-isothiocyanate (FITC) diluted 1:400 in 20% goat serum/PBS for 1 h at room temperature, washed with PBS and counterstained with PI (0.1 mg/ml) in the dark (30 min). Coverslips were mounted in mowiol (2.4 g mowiol 4–88 per 6 ml glycerol and 6 ml H2O with 0.6% 1,4 Diazabicyclo[2.2.2]octane (DABCO)) on glass slides and the percentage of cells that stained positive for BrdU incorporation determined using fluorescence microscopy. In parallel experiments (n ¼ 6), LnCaP cells (4 105) were seeded onto collagen-coated coverslips and grown in RPMI 1640/10% FCS (5% CO2, 371C). Lycopene (0.5–15 mM) was added to the medium on the next day and after 48 h cultures were treated with 10 mM BrdU for 4 h and processed using the methodology described above.

Statistical analysis Observations made in these experiments are not independent, thus analyses that account for their nonindependence were used. Using repeated measures regression, comparisons between concentration groups were made to allow for non-independence. The adjustment made to allow for this is to use ‘robustified’ standard errors.4 A heteroskedasticity-consistent covariance matrix estimator and a direct test for heteroskedasticity analyses were carried out using the statistical software STATE (Stata Corp LP, College Station, TX, USA). Clinical study – design The primary aim of the study was to determine whether PSA velocity (the rate of rise or fall of serum PSA) was altered by lycopene supplementation. A response was defined as occurring if PSA velocity post-intervention was decreased compared to the pre-intervention value. Calculations indicate that a sample size of 40 patients, followed up for 1 year would be sufficient to measure at least a 30% response rate to within 14% certainty (based on a 95% confidence interval (95% CI)). Patients Having gained local ethical committee approval, patients (n ¼ 41) were recruited into the study. All patients had been diagnosed with CaP after biopsy and had localized disease at diagnosis. None of these patients were on active treatment beyond careful clinical surveillance and serial analysis of serum PSA levels, the pretreatment values of which had to have an established positive gradient over time (Table 1). No patient had received hormonal therapy or any other treatment known to affect PSA in the year preceding study entry, and none were taking regular supplements of lycopene, vitamin C, flavonoids or carotenoids.


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Table 1 Patient information Mean values Age: 73 years Gleason score: 6 Time since diagnosis: 4 years PSA at diagnosis: 23.3 ng/ml Primary treatment

No.

External beam radiotherapy Watchful waiting/active surveillance Anti-androgen therapy Surgery

18 13 2 4

Abbreviation: PSA ¼ prostate-specific antigen.

Treatment Patients were given 10 mg lycopene per day (two tablets of Lycoplus – each tablet contains 5 mg lycopene, 100 mg vitamin C, 1.25 mg vitamin E, 0.83 mg phytoene and phytofluene and 0.21 mg b-carotene). A dosage of 10 mg lycopene was selected, as there is some evidence that enteric absorption does not increase with higher doses.5

Figure 1 Representative photomicrograph of PECs. Following ‘7day plus 48 h’ culture on coverslips (as described in Materials and methods), adhered cells were fixed with 3.7% formaldehyde (in PBS) and visualized using phase-contrast microscopy. Scale bar E20 mm.

Statistical analysis Linear regression was used to calculate pre- and posttreatment rates of PSA increase. As, during proliferation, CaP and PSA exhibit log-linear growth, PSA data were log transformed before regression analysis. A paired Wilcoxon sign-rank test was used to compare slopes of log PSA against time in patients pre- and post-treatment. Based on these slopes, pre- and post-treatment PSA doubling times were calculated as log 2/slope.

Results Lycopene inhibits DNA synthesis in PECs Primary cultures of human PECs were prepared from surgically removed prostate tissues, seeded onto collagen-coated glass coverslips; colonies each contained several hundred cells after 7–10 days (Figure 1). To determine whether lycopene might alter DNA synthesis in primary PECs, cultures were exposed to different concentrations of lycopene (1–20 mM), and after 48 h, 10 mM BrdU was added to the medium of each culture. BrdU incorporation into nuclei was visualized by staining with a mouse monoclonal anti-BrdU antibody followed by a goat anti-mouse antibody coupled to FITC (Figure 2). Cells were counterstained with PI and the percentage of cells (Xn ¼ 500) that incorporated BrdU into their nuclei (%BrdU) established using fluorescence microscopy. In the presence of lycopene, clear doserelated reductions in the proportion of FITC-stained (green) nuclei to PI-stained nuclei (red) were observed (Tables 2 and 3; Figure 3a and b). Figure 3 shows the effects of 48-h treatment of primary PECs with lycopene on %BrdU incorporation. In control cultures, %BrdU incorporation in primary PECs (7-day-old plus 48 h incubation) was 53.777.2% (Figure 3). Treatment of PECs with lycopene for 48 h at concentrations of 1, 5, 10, 15 or 20 mM induced dose-related reductions in %BrdU incorporation of 48.973.6, 37.975.4, 32.874.6, 25.673.4 or 23.376.2%, respectively. A similar dose-related reduction in %BrdU incorporation was observed in androgen-

Figure 2 DNA synthesis: photomicrographs showing the concentration-related effect of lycopene on incorporation of BrdU in primary PECs. PECs cultured for 7 days in serum-free PrEBM medium and incubated for 48 h in the absence (panel rows 1 and 2) or presence of 5 mM (panel row 3), 10 mM (panel row 4) or 15 mM (panel row 5) lycopene before labeling with BrdU (4 h). Cultures on glass coverslips were then fixed and co-stained with anti-BrdU antibodies/FITC (panels 1a–5a (green), to visualize BrdU incorporation and DNA synthesis) and counterstained with PI (panels 1a–5a (red)) to facilitate visualization of total nuclei). Magnification 40.

dependent LnCaP cells. From six separate experiments, a control %BrdU of 36.277.2% was observed. Treatment of LnCaP cells with lycopene for 48 h at concentrations of 5, 10 or 15 mM resulted in dose-related reductions in %BrdU incorporation of 28.177.5, 27.578.7 and 22.378.6%, respectively. Prostate Cancer and Prostatic Diseases


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Table 2 Comparisons with control group (medium only) Lycopene concentration (mM)

Mean difference in % cells

95% CI

P-value

4.83 15.78 20.93 28.07 30.38

4.24 to 13.91 3.11 to 28.46 6.91 to 18.76 20.22 to 35.92 18.62 to 42.15

0.23 0.02 0.001 o0.001 0.001

1 (n ¼ 6) 5 (n ¼ 6) 10 (n ¼ 6) 15 (n ¼ 4) 20 (n ¼ 3)

Abbreviation: CI ¼ confidence interval.

Table 3 Comparisons with 1% THF medium Lycopene concentration (mM)

Mean difference in % cells alive

1 (n ¼ 6) 5 (n ¼ 6) 10 (n ¼ 6) 15 (n ¼ 4) 20 (n ¼ 3)

3.27 7.68 12.83 19.97 22.28

95% CI 12.45 3.37 4.35 9.92 9.33

to to to to to

5.92 18.73 21.31 30.01 35.23

P-value 0.40 0.13 0.003 0.004 0.007

Abbreviations: CI ¼ confidence interval; THF ¼ tetrahydrofuran.

Clinical study Four patients (9%) who had been recruited to the study withdrew within 6 weeks of commencing due to inability to conform to the study protocol. None of these withdrew due to rapid progression or adverse events. The remaining 37 patients continued with the treatment regime for an average period of 10.4 months, undergoing monthly review and serum PSA estimates. Six patients developed disease progression (local symptoms or increasing PSA rise) leading to their withdrawal from the study and intervention according to best current medical practice (Table 4). There was no reported toxicity in any patient beyond discoloration of feces. Regression slopes of (log) PSA vs time decreased in 26/37 (70%, 95% CI: 53–84%) of the patients after supplementation and in eight cases (21%) the post-treatment slope was negative. For these latter patients, the average fall in PSA was equivalent to 2% over 28 days (i.e. an average slope (per day) of 0.000713). The Wilcoxon rank-sum test showed a statistically significant decrease in slope overall (P ¼ 0.0007). Analysis of the PSA doubling time (pre- vs post-treatment) showed a median increase after supplementation for 174 days; however, this was not statistically significant (P ¼ 0.18). Separate analysis of the two largest patient subgroups, those who had previously received external beam radiotherapy (n ¼ 17) and those who had always followed an active surveillance program (n ¼ 13), revealed no difference between these groups in terms of the numbers of patients whose slope decreased and whose slope became negative with supplementation, although given the small numbers direct statistical comparison is inappropriate (Table 5).

Discussion The morbidity and mortality of CaP varies across the globe. It was established many years ago that populations who migrate from low-risk countries, for example, Japan and Poland, to high-risk countries, such as the USA, have an increased risk of developing the disease.6 Prostate Cancer and Prostatic Diseases

Figure 3 Concentration-related effects of lycopene on %BrdU incorporation in PECs. PECs cultured for 7 days in serum-free PrEBM medium were exposed for 48 h, as indicated, to lycopene before labeling with BrdU (4 h). Cultures on glass coverslips were fixed and co-stained with anti-BrdU antibodies/FITC together with PI and the percentages of prostate cells (X500) that incorporated BrdU were determined. The results show the mean7s.d. for independent experiments (n ¼ 6). In independent experiments, the effects of THF (in which lycopene was dissolved before addition to cultures) on %BrdU incorporation into PECs were examined at 10-mM equivalent (45.677.3%; n ¼ 6) and at 15-mM equivalent (40.170.5%; n ¼ 4). Table 4 Patients progressing Patient no.

Previous treatment

Reason for removal

Treatment started

1 2

EBRT Anti-androgen therapy Surgery EBRT EBRT EBRT

Bothersome LUTS PSA rise

Bicalutamide Bicalutamide

PSA rise Acute urinary retention PSA rise PSA rise

Bicalutamide TURP Flutamide LHRH agonist

3 4 5 6

Abbreviations: EBRT ¼external beam radiotherapy; LUTS ¼ lower urinary tract symptoms; PSA ¼ prostate-specific antigen; TURP ¼ transurethral resection of prostate.

This has led to epidemiological studies investigating the link between many factors that may explain these alterations in risk, including the role of dietary changes. The oxidative damage of cellular protein, lipid and DNA has long been regarded as a possible mechanism of the


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Table 5 Separate analysis of RT and WW subgroups

RT WW

Decrease in regression slopes of (log) PSA against time

Post-treatment PSA decrease

76% (13/17) 61% (8/13)

12% (2/17) 38% (5/13)

Median PSA doubling time 211.4 days (interquartile range – 39.6 to 603.4 days) 604.4 days (interquartile range – 834.9 to 292.2 days)

Abbreviations: PSA ¼ prostate-specific antigen; RT ¼radiotherapy; WW ¼ watchful waiting.

development of cancer.7 Dietary antioxidants, therefore, may provide protection of DNA and membrane lipids from oxidative damage and thus it is not surprising that there has been a great deal of interest into the chemoprevention effects of these agents on cancer. It was data from the Health Professionals Follow-Up study that first described an inverse relationship between the intake of lycopene and the risk of CaP.8 Further studies have shown that a similar relationship exists with lycopene levels in the plasma9 and that this may be ‘more potent’ when related to sporadic rather than hereditary disease.10 Lycopene is one of the most powerful antioxidants of the carotenoid family leading to a great deal of interest in this compound. However, subsequent experimental data has suggested that any ‘anti-cancer’ properties of lycopene may involve alternative mechanisms such as the induction of gap-junction cellular communication through the increased synthesis of connexin 43,11,12 a stimulated increase in cellular differentiation,13 inhibition of cell-cycle progression14 and reductions in circulating levels of insulin-like growth factor-1 (IGF-1);15 high levels of IGF-1 are associated with an increased risk of developing CaP.16 More recent studies have also described a decrease in local expression of IGF-1 and androgen signaling in the lateral prostatic lobes of young rats supplemented with 200 p.p.m. lycopene for up to 8 weeks17 and a lycopene-driven downregulation of 5-a reductase and consequent reduced steroid target gene expression together with a similar effect on the expression of prostatic IGF-1 and IL-6 gene expression.18 This is interesting in terms of the potential effects on the behavior of both benign and malignant prostate disease. In vitro studies on immortalized CaP cell lines (both androgen-dependent and -independent) have already suggested that, at physiological concentrations, lycopene can significantly decrease cellular proliferation.19–21 Our results employing LnCaP cells confirm such findings and demonstrate the dose-dependent inhibitory effects of lycopene on DNA synthesis. However, LnCaP and other established CaP cell lines do not provide an accurate human model. Most available cell lines are metastatic in origin and have been cultured extensively under artificial conditions, as their removal from the original in vivo environment where an important paracrine relationship with stromal cells exists. Indeed, not all studies have shown inhibitory effects of lycopene on the growth of prostate cells. Lycopene can increase urokinase plasminogen activator receptor expression while failing to inhibit cell proliferation or promote connexin 43 expression in studies using the PC-3 cell line.22 Extrapolating experimental data to normal prostate physiology and the initiation of tumors and their growth therein, therefore, is not necessarily relevant if only these models are used. Indeed, results from animal model experiments have disappointingly shown little effect of dietary lycopene

supplementation, although positive effects have been described from the use of whole-tomato extract supplementation.23 While such observations are suggestive of alternative ‘anti-cancer’ agents in tomatoes, it lends weight to experimental results of Pastori et al.,19 who demonstrated a significant synergistic effect between lycopene and vitamin E in inhibiting PC3 cell proliferation in culture; a similar effect was described more recently in LnCaP cells.24 Tomatoes, and indeed the oleoresin tablets used in this phase II clinical study, contain a number of additional antioxidants, and although lycopene is by far the most abundant, a role for combination effects cannot be excluded.25 In this study, we have used primary PEC cultures derived from patients undergoing surgery and demonstrated that lycopene significantly reduces DNA synthesis in these cells consistent with previous work demonstrating an inhibitory effect of lycopene alone. These results reinforce the validity of performing clinical trials using dietary supplementation. Previous clinical trials have shown that lycopene supplements (30 mg/day), either in the form of tomatobased pasta sauce or oleoresin tablets, for a short period before radical prostatectomy does increase lycopene concentrations in prostate tissue and can decrease the positive margin rate, cancer volume, oxidative DNA damage and serum PSA.26,27 Subsequent analysis provided in vivo evidence that these effects related to tomato sauce consumption may be a result of an increase in apoptotic cell death.28 Furthermore, men with metastatic CaP have been shown, in a randomized study, to have an improved outcome on many fronts, including diseasespecific mortality, when treated with androgen deprivation by orchidectomy with the addition of lycopene supplementation (2 mg b.d.) compared to those treated with orchidectomy alone.29 Interestingly, a recent study examining the effect of a dietary supplement (‘verum’ – containing numerous potentially active compounds, including carotenoids, selenium and phyto-estrogens) in a group of patients not dissimilar to those included in this study describes a significant reduction in serum-free PSA but not in total PSA nor any increase in total PSA doubling times.30 The period of supplementation was only 6 weeks; however, this was a randomized, doubleblind placebo-controlled study.30 The clinical study described here reinforces the positive findings of these previous trials and demonstrates an ongoing and real effect upon serum PSA and perhaps therefore possibly on the behavior of established CaP in men receiving no other form of treatment. Indeed, while the overall effect was to demonstrate a statistically significant shallowing of the gradient of PSA, there was also a large increase in estimated PSA doubling time. Although this increase did not reach statistical significance, it must be remembered that the eight patients who had the best responses to lycopene supplementation (with an actual fall in their Prostate Cancer and Prostatic Diseases


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serum PSA) were excluded from that analysis, as this would have introduced a number of results of infinity to our calculations. Had these patients been included with even a nominal doubling of their PSA doubling time, this result would also have been significant at the 95% level. These studies show, for the first time, that lycopene can inhibit DNA synthesis in primary PECs in vitro suggesting that lycopene may play an important inhibitory role in their growth; this might be critical in benign and malignant prostate disease processes. Our pilot clinical study lends weight to the possibility that dietary supplementation with whole-tomato lycopene might slow disease progression in men with CaP. However, this pilot study is limited in that although the numbers included provide sufficient statistical power to draw conclusions, the patients enrolled before starting supplementation represent a wide spectrum drawn from what is a diverse disease having a varied clinical course. Furthermore, variations in base-line dietary habits are not included, although patients taking either supplements or extra tomatoes were not recruited. Dietary questionnaires in any case suffer significant reliability problems. As we were looking for evidence of possible disease modification, we deliberately did not use generally accepted oncologic response criteria (normalization or 50% drop in PSA). Our feeling was that to expect a complete response using only a low level of dietary supplement was an unreasonable expectation. Our results use PSA levels as a surrogate marker of disease activity, which may not be valid in patients treated with hormonally active compounds. Although unlikely, if lycopene acts in an anti-hormonal manner, it is possible that some of the PSA effect seen could be due to its direct action on the PSA promoter gene, which is known to have an androgen-response element. Only long-term clinical comparisons would clarify this. Despite such drawbacks, our study raises the possibility that (whole-tomato) lycopene supplementation may mean that periods of watchful waiting can be extended, thus delaying the inevitable side effects of hormone manipulation. While this pilot study might suggest that this may be the case, a large randomized, placebocontrolled study is clearly needed.

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