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Effect of lycopene on androgen receptor and prostate-specific antigen velocity

ZHANG, Xin; WANG, Qi; Barber, Neil; CHEN, Xiao

doi: 10.3760/cma.j.issn.0366-6999.2010.16.014
Original article
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SDC

Background There is increasing interest in the role of dietary factors in both the development and behaviour of prostate cancer. This study was carried out to evaluate the impact of the dietary factor lycopene on DNA synthesis, activity and expression of the androgen receptor gene element in prostate LnCaP cells and to report our pilot phase II study investigating its effect on prostate-specific antigen velocity over one year.

Methods LnCaP cells were grown with or without different concentrations of lycopene or tetrahydrofuran (THF solvent) added to the culture medium for 48 hours. DNA synthesis was measured by the incorporation of bromodeoxyuridine (Brdu) into DNA during a 4-hour pulse, followed by immunostaining and visualization of stained cells using fluorescence microscopy. A transient transfection of a plasmid DNA recombinant containing an androgen receptor element-luciferase (ARE-Luc) report gene into LnCaP cells was developed and the impact of different concentrations of lycopene on the androgen receptor element was reflected by quantitative analysis of the luciferase enzyme function. Expression of the androgen gene was also studied by Western blotting. The phase II pilot study patients (n=41) previously diagnosed with prostate cancer were enrolled and given lycopene supplement, 10 mg per day, and response was measured by observing changes in the plasma prostate-specific antigen (PSA) levels.

Results The addition of 0.5 μmol/L, 5 μmol/L, 10 μmol/L and 15 μmol/L of lycopene was shown to inhibit cell growth by 2.66%, 4.29%, 3.73% and 13.66%, respectively, compared with the THF solvent control samples (P=0.015). As compared with the RPMI1640 medium group, cell proliferation in the presence of 5 μmol/L, 10 μmol/L, and 15 μmol/L lycopene was inhibited by 8.12%, 6.33% and 12.00%, respectively (P=0.024). We showed for the first time that lycopene inhibited the activity of the androgen receptor gene element in a dose-related manner. Inhibition was seen in the transcription of the luciferase construct and confirmed by androgen receptor element expression assayed by Western blotting. 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 eight patients, the average fall in PSA was equivalent to 2% over 28 days (i.e. an average slope/d of -0.000 713). The Wilcoxon rank-sum test showed an overall statistically significant decrease in slope (P=0.0007). Analysis of the PSA doubling time (pretreatment vs. posttreatment) showed a median increase after supplementation for 174 days; however, this was not statistically significant (P=0.18).

Conclusions Lycopene as an antioxidant dietary factor could significantly inhibit DNA synthesis in a dose-dependent pattern; the result revealed lycopene might inhibit androgen receptor gene element activity and expression. Dietary lycopene may play an important role in prostate cancer cell proliferation and further supports a large randomized study into the role of lycopene supplementation in malignant prostate disease.

Department of Urology, Beijing Cancer Hospital & Peking University School of Oncology, Key Laboratory of Carcinogenesis and Translational Research of Ministry of Education, Beijing 100142, China (Zhang X and Chen X)

Department of Urology, Beijing Xuanwu Hospital, Capital Medical University, Beijing 100053, China (Wang Q) Department of Urology, King's College Hospital, London, UK (Neil B)

Correspondence to: Dr. CHEN Xiao, Department of Urology, Beijing Cancer Hospital & Peking University School of Oncology, Beijing 100142, China (Tel: 86–13701215303. Email: bjdxzlyy@sina.com)

This study was supported by a grant from the Beijing Science and Technology Committee (No. 2004-B-01).

(Received March 17, 2010)

Edited by SUN Jing

Prostate cancer is the second leading cause of death from cancer in men in the Western world.1 Morbidity is also increasing sharply in China. In recent years there has been increasing interest in the possible role of dietary factors in terms of the development and progression of cancers, including prostate cancer. Thanks to mounting circumstantial epidemiological and scientific data, although good clinical data is lacking. The role of oxidative damage to cellular proteins, lipids and DNA has been proposed as a possible mechanism of the evolution of cancer.2 Lycopene is an important and very powerful dietary antioxidant, which may provide protection against oxidative damage and thus interfere with tumour development and progression. Lycopene is a member of a group of natural pigments known as carotenoids. It can be synthesized by both plants and micro-organisms and is widely found in the environment, giving colour to a number of plant species. For humans, lycopene is found P in a relatively narrow range of foods and the principal source of dietary lycopene for most people is tomatoes.3 In the Western world 85% or more of dietary lycopene comes from tomatoes and in the UK the average daily intake is about 1 mg which is lower than that estimated for the USA by about five times.4 In the human body, dietary lycopene has been shown to concentrate in a number of specific organs, including the liver, prostate and adrenal glands. A number of epidemiological studies have shown an inverse relationship between dietary lycopene intake and the risk of prostate cancer.

Previous studies revealed that excessive and abnormal stimulation of prostate epithelial cells by androgen are an important prostate cancer risk factor.3 Current studies prove androgen has a potential effect on prostate cell proliferation and differentiation, as well as apoptosis. Androgen signals cells through a specific androgen receptor (AR). Research has shown that expression of an AR gene element was much higher in hormone refractory prostate cancer patients. Therefore, reduced expression of the AR gene element could be a potential treatment for prostate cancer.

Although many researchers have revealed that a higher level of plasma lycopene is associated with lower morbidity from prostate cancer, most studies up to now have been epidemiological studies. Therefore, the effect of lycopene on the proliferation of prostate cancer cells is not yet well understood. In this study, the effect of lycopene on the synthesis of DNA in LnCaP cells is determined and, for the first time, the ARE-Luc reporter gene was used to investigate lycopene regulation of gene transcription and the translation of the androgen receptor gene product. We report for the first time that lycopene can inhibit activity and expression of the androgen receptor gene element. As a result of these preliminary investigations, we proceeded to a small-scale study on patients (n=41) with CaP to investigate whether supplementary dietary lycopene would retard the rate of progression of the disease as reflected by changes in serum PSA levels. Results are to some extent satisfactory.

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METHODS

Lycopene solution

Lycopene powder (Sigma-Aldrich, USA) was purchased and kept at -70°C. Lycopene (1 mmol/L) stock was prepared in tetrahydrofuran (THF) in the dark room just before use.

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Cell culture

After seven days of growth, cultures were placed in fresh RPMI 1640 medium (Cambrex Company, Belgium) and different amounts of lycopene stock solution (1 mmol/L) were added to each well, resulting in final lycopene concentrations of 0.5 μmol/L, 5.0 μmol/L, 10.0 μmol/L and 15.0 μmol/L. The highest amounts of THF (Sigma-Aldrich, USA) used to dissolve lycopene (1%) were added to separate cultures to provide control populations of cells.

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DNA synthesis assay

Forty-eight hours after adding the lycopene, 20 mmol/L of 5-bromodeoxyuridine (Brdu) (Sigma-Aldrich) was added to each well and cultures incubated at 37°C for four hours. The cells were fixed in 4 % formaldehyde in PBS for 20 minutes at room temperature and subsequently permeabilized with 0.2 % TritonX-100, 2 mol/L HCl and 100 mmol/L Tris-HCl buffer. Monoclonal anti-Brdu antibody (Amersham Biosciences, UK, 1:25 diluted in 20% goat serum in PBS) was added to each sample and incubated for 1 hour at room temperature. Goat anti-mouse-FITC antibody (Southern Biotechnology, UK) diluted 1 to 400 in 20% goat serum and PBS was then added as the secondary antibody and samples incubated for one hour at room temperature. Propidium iodide (PI) (Amersham Biosciences, 0.33 ng/ml) was then added for 15 minutes to stain DNA. Fluorescence light microscopy was used to visualize stained cells which were scored first for PI under yellow fluorescence and then for BrdU under green fluorescence. The number of growing cells undergoing DNA synthesis was calculated as the percentage of Brdu stained cells.

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Construction of luciferase report gene and evaluation of androgen receptor gene element activity

The ARE-Luc (Androgen Responsive Element-Luciferase) report gene construct was donated by King's College Hospital, UK. The self-construct plasmid is shown in Figure 1. Lipofectin reagent for transfection was from the Invitrogen Company. The construct plasmid was transfected into the LnCaP cell line using the liposome-mediated method. We mixed 375 μl of the transfected plasmid (40 ng/μl) with 0.75 ml of optimem I (Sigma) and after 15 minutes added 2.5 ml Optimem I and 75 μl lipofectin. One ml of the above liquid was then added to the cultured cells. After transfection different concentrations of lycopene were added to the transfected LnCaP cells.

Figure 1.

Figure 1.

Cells were then lysed with 200 μl lysis reagent (Promega) and centrifuged at 12 000 rpm for two minutes. The upper layer liquid was transferred to different tubes for luciferase assay, protein kept at â20δC for BSA protein assay. One hundred μl of the luciferase assay substrate (Promega) was transferred into tubes and 20 μl samples of the above lysis liquid from cells treated with different concentrations of lycopene was added. Constructed plasmid contained luciferase enzyme gene and was translated into luciferase enzyme which could mediate a fluorescent light lumination reaction, change substrate into luciferase and the latter luciferase was counted by luciferase luminometer. Results were demonstrated as light intensity. Luciferase activity was normalized to protein concentration and compared to the lycopene concentration used in the cell culture to reflect androgen receptor gene activity.

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Western blotting for androgen receptor gene element

Lysates of all samples were also taken for Western blot to evaluate transfected androgen receptor gene element expression.

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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 level.

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Patients

We received ethnic committee approval and 41 patients were recruited into the study. They had a mean age of (73±13) years and Gleason score was 6. PSA at diagnosis was (23.3±9.2) ng/ml. All patients had been diagnosed with CaP after biopsy and had localized disease as a diagnosis. None of these patients were on further active treatment beyond careful clinical surveillance and serial analysis of serum PSA levels, the pretreatment values had increased over time. In the year preceding study entry, no patient had received hormonal therapy or any other treatment known to affect PSA, and none were taking regular supplements of lycopene, vitamin C, flavonoids or carotenoids.

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Treatment

Patients were given 10 mg lycopene per day (two 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 β-carotene). A dosage of 10 mg lycopene was selected, as there is some evidence that enteric absorption does increase with higher doses.

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Statistical analysis

Experiment values were expressed as means ± standard deviation (SD). A Student's t test was used to compare the difference between the lycopene group of cells and the appropriate control cells incubated in medium alone or solvent. Linear regression was used to calculate pre- and post-treatment rates of PSA increase. A paired Wilcoxon sign-rank test was used to compare slopes of log PSA against time in patients pre- and post-treatment. Based on those slopes, pre- and post-treatment PSA doubling times were calculated as log2/slope. P value less than 0.05 was considered statistically significant.

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RESULTS

DNA synthesis

LnCaP cells grew rapidly in medium with appropriate supplements and reached confluence after a period of 7–10 days of culture. In initial experiments, cultures of LnCaP cells were stained for DNA synthesis using BrdU. PI stain was used to visualize all cells. In the RPMI-only medium group, approximately 40%-60% of cells that were stained by PI also incorporated BrdU into their DNA during a 4-hour pulse; demonstrating that these populations of cells were undergoing rapid growth (Figure 2). The addition of lycopene to the medium caused a dramatic and dose-dependent inhibition of the incorporation of BrdU into DNA and reduced the number of and proliferation of these cells (Figure 3). Analysis of the results demonstrates a dose-dependent effect of lycopene at the concentrations used in this study.

Figure 2.

Figure 2.

Figure 3.

Figure 3.

The addition of 0.5 μmol/L, 5 μmol/L, 10 μmol/L and 15 μmol/L lycopene inhibited cell growth by 2.66%, 4.29%, 3.73%, 13.66% (P=0.015) respectively compared with solvent 1% THF control samples. Compared with the RPMI 1640 group, the proliferation of the group with 5 μmol/L, 10 μmol/L and 15 μmol/L lycopene was inhibited by 8.12%, 6.33% and 12.00%, respectively (P=0.024) (Figure 4).

Figure 4.

Figure 4.

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Androgen receptor gene element activity

Luciferase activity, measured as light intensity, indicates the amount of luciferase enzyme produced in cells transfected with the ARE-Luc gene and represents transcription and translation of the AR gene. Light intensity readings were compared among the groups treated with different lycopene concentrations and between groups treated with lycopene and the THF solvent. Light intensity readings were lower in each lycopene treated group compared with the relevant control group. Luciferase activity diminished with lycopene concentration increase in a dose related manner (Figure 5).

Figure 5.

Figure 5.

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Androgen gene element expression

Androgen receptor element expression was inhibited in a dose-dependent manner. When lycopene concentration reached 15 μmol/L, androgen receptor element expression was thoroughly inhibited (Figure 6).

Figure 6.

Figure 6.

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Clinical study

Four patients (9%) who had been recruited to the study withdraw within six 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 progressive disease (local symptoms or increasing PSA levels) leading to their withdrawal from the study and intervention according to best current medical practice (Table 1). There was no reported toxicity in any patient beyond discoloration of feces.

Table 1

Table 1

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 change in slope/d of -0.000 713). The Wilcoxon rank-sum test showed a statistically significant decrease in slope overall (P=0.0007). Analysis of the PSA doubling time (pre-treatment vs. post-treatment) showed a median increase after supplementation for 174 days, however, this was not statistically significant (P=0.18). We did a separate analysis of the two patient subgroups, those who had previously received external beam radiotherapy (n=6) and those who had always followed an active surveillance program (n=10). We found no difference between these groups in terms of the number of patients whose slope decreased and whose slope became negative with supplementation; although given the small numbers direct statistical comparison is inappropriate (Table 2).

Table 2

Table 2

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DISCUSSION

The morbidity and mortality of prostate cancer differs in different areas in the world but is strongly associated with dietary factors. Populations who migrate from low risk countries, for example Japan and Poland to the USA, suffer an increase risk of developing the disease.5 In many countries, and particularly in the USA, this has led to epidemiological studies investigating the link between diet and all cancers; including that of the prostate gland. It was the data from the Health Professionals Follow Up study that first described an inverse relationship between the intake of lycopene and the risk of prostate cancer.6 Further studies have shown that a similar relationship exists with lycopene levels in the plasma.7 The oxidative damage to cellular proteins, lipids and DNA is regarded as a possible mechanism in the evolution of cancer.8 Dietary antioxidants, therefore, may provide protection of DNA and membrane lipids from oxidative damage and lycopene is one of the most powerful antioxidants in the carotenoid family.9,10 However, “anti-cancer” properties of lycopene may also be mediated through alternative mechanisms; including the induction of gap-junction communication between cells through the increased synthesis of connexin 43, an effect which has also been described.11

Although in recent years, epidemiological studies have shown that high level of dietary lycopene could reduce risk of prostate cancer, research into the function of lycopene is far from conclusive. Some in vitro studies on human prostate carcinoma cell lines PC3, LnCaP, and DU145 have already shown that, at physiological concentrations, lycopene can significantly decrease proliferation in prostate cancer cells.12–16 But inhibitory function in these studies has not yet been clarified.

Clinical trials have shown that lycopene supplements (30 mg per day), either in the form of tomato based pasta sauce or oleoresin tablets, for one month prior to radical prostatectomy can decrease the positive margin, cancer volume and PSA levels.17–19 In a randomized study men with metastatic prostate cancer have been shown to have an improved outcome on many fronts, including disease specific mortality, when treated with androgen deprivation by orchidectomy with lycopene supplementations (2 mg bid) versus those treated with orchidectomy alone.20

The clinical study described here reinforces the positive findings of previous clinical trials and demonstrates an ongoing and real effect upon serum PSA, and may represent a change in the biology of established CaP tumors in men who are receiving no other forms of treatment. The overall effect was to demonstrate a statistically significant shallowing of the gradient of PSA, and there was a large increase in established 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 serum PSA) were excluded from that analysis, as this would have introduced a number of results of infinity to our calculations.

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 number included provided sufficient statistical power to draw conclusions, the patients enrolled before starting supplementation represent a group with diverse disease having varied clinical courses. Furthermore, variations in base-line dietary habits are not included, although patients taking either supplements or extra tomatoes were not recruited. Results from dietary questionnaires may contain significant reliability problems.

This study revealed that besides an anti-oxidant mechanism, lycopene may also act in an anti-hormonal manner, through inhibition of the androgen receptor element that led to the reduction of PSA velocity. It would suggest that a large, long running, randomized trial into the possible role of dietary supplementation of lycopene in both benign and malignant prostate diseases would be an appropriate course of action.

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Keywords:

lycopene; prostate cancer; LnCaP; androgen receptor element

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