Chemopreventive Effects of Tea in Prostate Cancer: Green Tea vs. Black Tea

Abstract

1 Introduction

The evidence for chemoprevention by any bioactive substance is drawn from a combination of epidemiological, animal and basic mechanistic studies as well as limited amounts of evidence from human intervention studies. This is particularly true for tea which is the second most commonly consumed beverage in the world after water. The vast majority of the tea consumed in the world is black tea (BT), comprising approximately eighty percent of all tea consumed [1]. Nonetheless, the scientific evidence for the chemopreventive activities of green tea (GT) is far more extensive than that available for BT. The key chemical differences between BT and GT result from post-harvest processing. During GT tea production the endogenous oxidase enzymes in tea leaves are inactivated by heating so that the green tea polyphenols (GTPs), including epigallocatechin gallate (EGCG), are preserved. In BT, natural oxidation after harvesting results in polymerization and formation of theaflavins and thearubigins with small amounts of EGCG remaining in BT [1]. After ingestion, both BT and GT undergo fermentation by normal colonic flora also known as the microbiome. This metabolism results in the formation of phenolic acids, which may be active in chemoprevention. Therefore, the key differences between GT and BT are that GTPs are found in higher concentration in the circulation after consumption of GT compared to BT. On the other hand, the phenolic acid levels in the circulation are higher after consumption of BT by comparison to GT. This article will review in detail the available epidemiological and human clinical studies, as well as animal and basic mechanistic studies on GT and BT supporting a chemopreventive role in prostate cancer (CaP). This review will also highlight the importance of investigating the bioavailability and metabolism of polyphenols in CaP chemoprevention.

2 Prostate cancer

Prostate cancer is the second most frequently diagnosed cancer and the sixth leading cause of cancer death among men worldwide, with 914,000 new cases and 258,000 deaths in 2008 [2]. More than half of these cases and deaths are expected to occur in more developed countries [2]. The highest country-specific age-standardized incidence rate (per 100000 population) in 2002 was 124.8 in the USA, and the lowest in Bangladesh (0.3), in China (1.6) and in India (4.4) [3]. Much of the observed geographic variation in CaP incidence may be due to differences in prostate-specific antigen (PSA) testing, and the ability to detect latent CaP [2]. However, differences in the use of PSA testing cannot explain all of the international variation, since there was already more than a 50-fold difference in international CaP incidence rates across countries in 1980 before the PSA test was introduced [3]. This indicates that environmental factors have a substantial role in determining cancer risk. In support of an environmental influence it was observed that men born in Japan and immigrating to Hawaii assimilated the host country's cancer rate both in their lifetime and in succeeding generations [4]. Diet is probably one of the most important environmental risk factors for CaP. The CaP incidence is low in Asian countries, and one possible explanation is the high intake of soy, tea, fish, fruits and vegetables and the reduced intake of red meat and fatty foods by comparison to the Western Diet [5]. However, the incidence of CaP is increasing rapidly in Asian countries due to the rapid introduction of a Western lifestyle, especially in urban centers [5]. CaP is typically diagnosed in men over 50 years of age and the rate of growth and progression is typically slow, which makes CaP an ideal disease in which to study the utility of preventive strategies, including those resulting from changes in nutrition and lifestyle [6]. Due to the fact that most CaP treatments carry the risk of side effects, there is an increasing trend for men diagnosed with less advanced CaP to choose expectant management, also called active surveillance, which provides additional opportunities to study non-toxic chemopreventive strategies such as GT.

3 Green and black tea

Tea is the most commonly consumed beverage second only to water in the world. All different varieties of tea including white, green, oolong, black and pu-erh teas are manufactured from the leaves of Camellia sinensis. For the production of GT the leaves are heat-treated to retain the typical polyphenols, also known as flavan-3-ols, including (-)-epigallocatechin (EGC), (-)-epigallocatechin-3-gallate (EGCG), (-)-epicatechin (EC), and (-)-epicatechin-3-gallate (ECG) [1]. For the production of BT the leaves undergo fermentation in moist and warm conditions [1]. Endogenous enzymes enhance the formation of polymers such as theaflavin and thearubigen. Therefore, BT contains a relatively small amount of monomeric polyphenols (Table 1). Other differences are the higher content of theanine and lower content of caffeine in GT [7]. Worldwide, 78 percent of tea produced is BT. By contrast GT comprises 20% of tea production and is the preferred form in China and Japan, as well as a few countries in North Africa and the Middle East. On the other hand, BT is consumed in European countries, the US, India, and many Arabic countries. The main tea producing countries are China, India, Kenya, Sri Lanka, Turkey, Indonesia, and Japan (Food and Agriculture Organization of the United Nations—Production FAOSTAT).

GT has been studied extensively for its chemopreventive activities with regard to CaP [8-10]. After evaluating all the evidence it becomes clear that there is a strong role for GT in chemoprevention of CaP, but much more research is needed on the chemopreventive potential of BT. More clinical research is needed for both GT and BT.

4 Animal studies

Convincing evidence exists showing that GT extracts (GTE) decrease tumor growth in a variety of animal models. However, there are very few animal studies using brewed GT, and BT has not been studied as often as GT. However, several investigations using BT extracts demonstrated an inhibition of CaP growth in mouse xenograft and a rat models [1;11;12]. The majority of mouse studies are summarized in the review by Johnson et al [8]. Mouse xenograft models used both androgen-dependent (LNCaP and CWR22Rv1) and androgen-independent (PC-3, LNCaP-R104, CWR22R) cell lines. Both demonstrated a decrease in tumor growth with administration of EGCG or GTPs (Table 2). We will highlight a few important studies. Two studies investigated whether inhibition of tumor growth by GT intervention depends on the stage of CaP development [13;14]. In the study by Harper et al. 0.06% EGCG in drinking water decreased tumor growth at 12 weeks but not at 28 weeks of age in transgenic adenocarcinoma of the mouse prostate (TRAMP) model by inhibiting proliferation, inducing apoptosis, decreasing androgen receptor, insulin-like growth factor-1 (IGF-1), IGF-1 receptor, phosphor-extracellular signal-regulated kinases (ERK) 1 and 2, cyclooxygenase-2 and inducible nitric oxide synthase (iNOS) [14]. The study by Adhami et al. demonstrated that treatment of TRAMP mice with 0.1% of GTE extended tumor-free survival significantly when the intervention was initiated prior to week 28. In addition, a decrease in IGF-1, IGFBP-3 and phosphatidylinositol 3-kinase (PI3K)/AKT/ERK was only observed when treatment was started prior to week 28 [13]. Another important mouse study explored the question of the GTE concentration necessary for chemoprevention in an androgen dependent xenograft model tumor model in nude mice [11]. The investigators determined that the administration of 0.05% of GTE in drinking water was associated with a significant extension in days to reach 1200 mm tumor volume but not at 0.01%. This was associated with a decrease in PSA, an increase in apoptosis (PARP, caspase, Bax, Bcl2) and a decrease in vascular endothelial growth factor (VEGF) [11]. However in a few mouse studies GT administration was not effective [15;16]. For example in the study by Zhou et al. an intraprostatic inoculation of male severe combined immune deficient (SCID) mice with the androgen-sensitive human LNCaP CaP cell line was used. Oral administration of brewed BT but not GT in drinking water demonstrated a significant decrease in tumor weight associated with decreased serum PSA [15]. China green and black leaf tea imported from Shanghai Tea Import was used at a concentration of 15 g of tea leaves per liter of water [15]. On a weight basis this is a much higher intake of BT than typical for humans. Another interesting xenograft mouse model included the subcutaneous injection of TRAMP-C1 cells with or without lipopolysaccharide (LPS) in C57/Bl mice [16]. 0.6% GTE (59% EGCG) was administered in drinking water starting three days prior to tumor injection. GTPs did not inhibit tumor growth. However, LPS-induced recruitment of inflammatory polymorphonuclear phagocytes (PMNs) significantly decreased tumor growth, which was inhibited by GTP administration [16].

Most mouse studies investigating the chemopreventive and therapeutic effects of GT were performed using the TRAMP model and using the chemical EGCG or GTEs. TRAMP mice express the rat probasin (PB)-SV40 early gene (T/t antigen) construct under prostate specific control of the minimal rat probasin promoter and display mild to severe hyperplasia of the prostate epithelium, resembling prostatic intraepithelial neoplasia (PIN) by 6-12 weeks of age. Between 10 to 16 weeks well-differentiated neoplasia is generally observed, between 18-24 weeks of age all of the mice will display primary tumors, and by week 30 will display metastases to distant sites [17]. Table 2 gives an overview of the TRAMP tea intervention studies. Most of the studies used GTE enriched in EGCG. The lowest effective concentration was 0.1% GTE in drinking water. In most studies GTPs were administered starting at 4 to 8 weeks of age. When GT was started at later points in time, the effect on tumor progression was decreased [13;18]. This could indicate that GT has more of a preventive effect earlier in transgenic tumor progression and less of a therapeutic effect once tumors are established. Moreover, not all investigations using TRAMP mice demonstrated protection. A study by Teichert et al. used 0.05% GTPs with unknown EGCG content and did not show a decrease in tumor weight [19]. Another study by Morey Kinney et al. administered orally 0.1% GTPs with 35% of EGCG and did not show a decrease in tumor progression [20]. In the same study no effect on DNA methylation in the prostate of TRAMP mice was found after administration of 0.1-0.6% GTPs in drinking water.

Few rat studies have been performed to investigate the chemopreventive effect of GT. One study by O'Sullivan et al. showed a decrease in tumor progression of 50% compared to water control but no decrease in tissue oxidative DNA damage marker, 8-hydroxydeoxyguanosine (8-OHdG). However manganese superoxide dismutase (MnSOD) protein expression was increased by the tea treatment [21].

Animal models clearly provide rapid answers by comparison to clinical studies, and serve a useful purpose in highlighting potential actions and interactions of candidate chemopreventive substances such as tea. However, there is some question as to how these studies relate to human benefit. Many of the studies are done with subcutaneous injection of tumor cells into an immune-impaired host and do not replicate the complex microenvironment of the human prostate gland. Nonetheless, the xenograft tumors are invaded by host immune and stromal cells and demonstrate tumor angiogenesis and elements of metastasis in appropriate model systems. Therefore, they do recapitulate elements of human CaP. Ultimately human intervention studies provide the best quality of evidence for the chemopreventive potential of tea.

5 Human epidemiological studies

As summarized in Table 3, twelve population studies, including five case-control and seven cohort studies, have been published evaluating the association of tea consumption with CaP. Among these population studies four evaluated the use of GT alone, two evaluated GT and BT, one evaluated only BT and five did not define the type of tea. The studies analyzing “tea” from Western countries presumably examine BT since this is the most common tea consumed in Western countries. Among the cohort studies, one GT study showed a protective effect in advanced CaP [22] and one BT study showed a protective effect in localized CaP associated with “tea” consumption [23]. Two GT cohort studies in Japan showed no association, one GT and BT cohort study from Hawaii in men with Japanese ancestry showed no effect and two cohort studies not defining the type of tea (Canada, USA) showed no effect (Table 3).

Among the case control studies one GT study from China showed a protective effect [24]. One tea study from Canada, most likely investigating BT, showed a protective effect and another study from Japan investigating GT and BT showed a trend to protective effect [25]. However there were two case control studies from Italy and Canada with “tea” not showing an association [26;27].

6 Human intervention studies

Data from two intervention studies in patients with localized CaP and two in patients with hormone refractory CaP have been published [33-36]. The first intervention study of localized CaP, performed in Italy, showed that GTPs are effective in delaying progression of premalignant lesions to CaP was [33]. 60 men with high-grade prostate intraepithelial neoplasia (HG-PIN) in the baseline biopsy were randomized to either 600 mg of a decaffeinated GTE containing 51.8% of EGCG or placebo daily. At the end of one year repeat biopsy samples were taken and men in the placebo group had a 30 percent incidence of CaP compared to only 3 percent in the group receiving the GTE [33]. Serum PSA showed a non-significant trend to decrease in GTE-treated men at 9 and 12 months. A significant improvement was observed for the international prostate symptoms score. GTE administration also reduced lower urinary tract symptoms, suggesting that GTPs might be helpful in treating benign prostate hyperplasia (BPH). A two year follow up assessment was published in 2008 in a letter to the editor of European Urology [37]. Only 9 participants from the placebo-arm and 13 from the GTE-arm underwent a third biopsy sample collection. Despite the high drop-out rate the two arms remained balanced and large enough for statistical analysis. Two further cancer diagnoses appeared in the placebo arm and one in the GTE-arm. This indicates that overall, even after suspension of the GTE treatment, the GT-arm experienced an almost 80% reduction in CaP diagnosis compared to the placebo group [37]. In the second smaller intervention trial with localized CaP 25 men diagnosed with stage I, II, or III CaP, who were scheduled for prostatectomy consumed 4 capsules of Polyphenon E daily containing a total daily dose of 800 mg of EGCG [34]. The average intervention time was 4-6 weeks. Only one patient reported mild nausea related to the Polyphenon E intake. No adverse effects on liver function were observed. A significant decrease in serum PSA, hepatic growth factor (HGF), vascular endothelial growth factor (VEGF), insulin growth factor 1 (IGF-1) and ratio of IGF-1 to insulin growth factor binding protein 3 (IGFBP-3) was found [34]. In addition two intervention studies from Canada and the US investigating hormone refractory CaP showed a very limited protective effect on CaP by consumption of GTE [35;36]. In one study 6g of a pulverized GT powder which contained sugar, citric acid and flavoring was administered [36]. However no information was provided about the GTP content of this GT product. Only one participant of 42 showed a decrease in PSA level. The second intervention study with hormone refractory CaP administered a dose of 250 mg of GTE containing 75% GTPs twice daily [35]. Nine patient of 15 consuming the GTE for more than 2 months had progressive disease and six showed an apparent slowing of disease progression with a slow rise in PSA serum level. However, there were no “responders” as per the traditional definition of a PSA drop of greater than 50%. There were no control groups in either of the two studies. Unfortunately no clear conclusion can be drawn from the studies of hormone refractory disease due to lack of information on the GTP content in the first study [36] and the use of a relatively low dose in the second study [35]. The results of the intervention studies support the findings from animal studies that GT is more effective in prevention and early stages of CaP and less effective in hormone refractory disease.

7 Bioavailability and metabolism of tea polyphenols

The limited bioavailability and extensive metabolism of tea polyphenols after absorption provides a challenge to their utilization for chemoprevention [38]. Tea polyphenols are mainly absorbed from the small intestine. The absorption is regulated by multidrug resistance-associated protein transporter (MRP) and monocarboxylate transporter (MCT) [38]. MRP1 is located at the basal membrane and MRP2 at the apical membrane. GTPs are taken up into the epithelial cells and metabolized to glucuronides, sulfates and methyl metabolites [39]. Both GTPs and metabolites can be transported to the intestinal lumen or into the vascular system [38]. In humans and mice the rate of conjugation depends on the chemical structure of the GTPs. Gallated polyphenols such as EGCG and ECG are found in the circulation in the free form whereas nongallated polyphenols circulate mostly in conjugated form [40]. In addition all GTPs undergo methylation catalyzed by catechol-O-methyltransferase (COMT) [41]. Non-gallated polyphenols are excreted through the kidney into the urine, whereas gallated polyphenols are not found in the urine and have been demonstrated to be excreted through the enterohepatic circulation [40].

Human prostate tissue concentrations have been determined by our laboratory in men who participated in a phase II tea intervention study at the University of California Los Angeles. Men consumed 6 cups of GT or BT 3-8 weeks prior to prostatectomy. The 6 cups of GT provided 571 mg of EGCG, 291 mg of EGC, 75 mg of EC and 89 mg of ECG daily. Tissue aliquots collected after prostatectomy from men consuming GT contained EGCG, 4″-O-MeEGCG and ECG (Table 5) [42]. No GTPs were found in tissue from men in the BT or control groups. Participants collected urine samples during the intervention. Urine samples from men consuming GT contained EGC, 4′-O-MeEGC and EC (Table 6). Urine samples from men consuming BT contained 100 fold lower amounts of the same GTPs. No GTPs were found in the control group consuming only water [42]. In a prior pilot study, also designed as a preprostatectomy trial, a Darjeeling BT was used, which contained higher amounts of GTPs [43]. In this human study no theaflavins were found in the human prostate after BT consumption. However, when administering a very high concentration of decaf. BT extract (5%) mixed into the diet to C57BL/6 mice we demonstrated that theaflavins can be absorbed into liver, prostate, small intestine and colon [43].

Due to the high rate of EGCG-methylation in human prostate tissue, we investigated the bioactivity of the methyl metabolite of EGCG. We demonstrated that methylation of EGCG decreased its pro-apoptotic and nuclear factor kappa B (NFκB) inhibitory activities [42]. Similar observations were made by other investigators, who found that methylation of EGCG and ECG decreased their proteasome-inhibitory activity [44]. As shown in Table 5 and ​and66 about 50% or more of EGCG and EGC found in human prostate and urine are in the methylated and less active form. Similar methylation rates were found in tissues of mice drinking brewed GT as drinking water (Figure 2) (unpublished data). GTP concentrations were similar in mouse and human tissues. However in human tissue only EGCG, 4″-MeEGCG and ECG were found, whereas the mouse tissue contained EC, EGC, 4′-MeECG, EGCG and 4″-MeEGCG. After the intragastric administration of 164 μmol/kg (equivalent to about 2.2 mg per 30 g mouse) EGCG to mice similar EGCG tissue concentration were found with (C_max) for EGCG (nmol/g) 0.09-0.2 in prostate, 0.03-0.1 in liver, 0.002-0.01 in lung, 45±14 in small intestine and 7.9±2.4 in colon [45]. A study by Meng et al. found EGCG and 4′4″-DiMeEGCG in liver, kidney and small intestine, plasma, urine and feces [46]. As substrates of methyltransferases, GTPs are able to inactivate methylating enzymes such as COMT and DNA methyltransferase (DNMT1). There is the potential that GT may inhibit carcinogenesis by limiting methylation of DNA and affecting the expression of proteins that stimulate proliferation [47;48]. The inhibition of DNMT1 activity by GTPs in cell culture has been demonstrated to lead to demethylation of the CpG islands in the promoter regions and the reactivation of methylation-silenced genes such as p16INK4a, retinoic acid receptor beta, O6-methylguanine methyltransferase, human mutL homolog 1, and glutathione S-transferase-pi (GSTp1) [48]. Since CaP is commonly associated with hypermethylation and silencing of GSTp1 it could be possible that GT intervention may assist in reactivation of GSTp1 [49;50] thus increasing its antioxidant activity and inhibiting tumor growth.

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Figure 2

Tissue concentrations of GTPs were determined in SCID mice inoculated s.c. with LAPC4 androgen-dependent prostate cancer cells. Drinking water was replaced with brewed green tea containing 0.07% GTPs freshly prepared every 2 days. 10 weeks after tumor cell inoculation mice were sacrificed and tumor, lung, liver and kidney tissue was collected and analyzed by high performance liquid chromatography with Coularray electrochemical detection (mean±std; n=5)[107].

Due to the limited absorption of GTPs and BT theaflavins, the GTPs remaining in the small intestine are transformed in the colon by the microflora to phenolic acids. In a study designed to examine the effects of GT and BT digestion products, an artificial colon containing human colonic microflora (TNO, Holland) was incubated with GT and BT concentrates [51]. The major phenolic acids found in the artificial colon content after GT and BT digestion were 3-methoxy-4-hydroxyphenylacetic acid, 4-hydroxyphenyl acetic acid, 3,4- dihydroxyphenylacetic acid, 3,3-hydroxyphenylpropionic acid, 2,4,6-trihydroxybenzoic acid [51].

The composition of BT varies strongly depending on the growing region and manufacturing conditions [32]. For example, Darjeeling tea contains higher amounts of EGCG compared to regular BTs [32]. Most in vivo BT intervention studies have analyzed plasma GTP concentrations. A study by Mulder et al. demonstrated that theaflavins are only minimally bioavailable [52]. 800 mg of theaflavin, equivalent to 30 cups of BT, was administered to two volunteers leading to a plasma concentration of 1 μg/L at 2 hours [52]. It is unlikely that theaflavin and thearubigins directly contribute to the chemopreventive effects of BT. Biological effects of BT consumption most likely are due to the GTPs and phenolic acids resulting from colonic metabolism. Therefore, in vitro evidence of potential molecular targets of BT theaflavins will not be discussed as this research is unlikely to be relevant to CaP chemoprevention. However, the bioactivity of the microflora products may be more relevant to the chemopreventive effects of BT. Further research is needed to determine whether the tissue concentrations of phenolic acids are sufficient to exhibit a chemopreventive effect. Preliminary research indicated that the IC₅₀ concentration to inhibit cell proliferation in HCT116 colon cancer cells at 75 μmol/L is much higher than the achievable tissue concentrations [51].

BT frequently is consumed in combination with a small amount of milk. The majority of investigations demonstrated that the addition of milk did not decrease the bioavailability of GTPs from BT [53;54]. However, one study by Reddy et al demonstrated a decrease of plasma GTPs when BT was consumed with milk [55]. The same study also demonstrated that the plasma antioxidant activity was not affected by milk [55]. Other studies showed mixed results on the effect of BT and milk combination on the antioxidant activity. The study by Kyle et al. 2007 demonstrated no decrease of the plasma antioxidant activity but the study by Ryan et al. demonstrated a decrease in the in vitro antioxidant of BT when combined with milk [54;56].

8 Molecular targets of GTPs in prostate cancer

Polyphenolic botanical extracts such as GTPs exert their effects on tumor growth through multiple mechanisms reviewed below.

9. Conclusion

Strong evidence from in vitro and in vivo animal studies supports the role of GT in CaP prevention. However, prior to assuming that these benefits translate to humans, several points need to be considered. First, as pointed out in this review, most of the in vitro studies do not take the pro-oxidant activity of EGCG at alkaline pH into consideration. With the elimination of the “hydrogen peroxide” effect, much higher concentrations of GTPs would be necessary to induce the same effects. Second, it appears that mouse tissue bioavailability of GTPs is considerably different from human tissue. Therefore the bioavailability in human tissue may be more limited and the potential of GTPs more limited.

Nonetheless, human population studies provide some supportive evidence for a decrease in risk of CaP associated with increased consumption of tea. Evidence is stronger for GT compared to BT. However, for the evidence to be convincing, population studies showing a beneficial effect of tea need to be replicated. The evidence from human clinical trials demonstrating a decrease in the rate of tumor progression from PIN to adenocarcinoma together with evidence of a decrease in serum markers of tumor progression provide strong support for the preventive actions of GT in localized CaP [33;34]. Further intervention studies are needed to demonstrate the effect of brewed tea, which is consumed in amounts relevant to the demonstrated intakes in epidemiological studies. Currently our group is conducting a phase II clinical trial investigating the effects of the consumption of 6 cups of GT or BT prior to prostatectomy on biomarkers of CaP. This trial will be completed in 2011 (Clinical Trial ID: NCI-2010-00973).

Overall, epidemiological and human clinical studies, as well as animal and basic mechanistic studies on GT and BT support a chemopreventive role in CaP with an emphasis on GT, but more research efforts at many levels is needed.

Footnotes

9 References