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Carcinogenesis vol.27 no.4 pp.833–839, 2006 doi:10.1093/carcin/bgi323 Advance Access publication December 29, 2005 Inhibition of CWR22Rn1 tumor growth and PSA secretion in athymic nude mice by green and black teas Imtiaz A.Siddiqui, Najia Zaman, Moammir H.Aziz, Shannon R.Reagan-Shaw, Sami Sarfaraz, Vaqar M.Adhami, Nihal Ahmad, Sheikh Raisuddin1 and Hasan Mukhtar Department of Dermatology, University of Wisconsin-Madison, WI 53706, USA and 1Department of Medical Elementology and Toxicology, Hamdard University, New Delhi, India To whom correspondence should be addressed. Tel: +1-608-263-3927; Fax: +1-608-263-5223; Email: [email protected] Cancer of the prostate gland (CaP), the most common invasive malignancy and a major cause of cancer related deaths in male population in the USA, is an ideal candidate disease for chemoprevention because it is typically detected in elderly population with a relatively slower rate of growth and progression. Many dietary phytochemicals are showing promising chemopreventive effects, at-least in pre-clinical models of CaP. Our published data in cell culture and animal studies, supported by the work from other laboratories, as well as epidemiological observations and case–control studies, suggest that polyphenols present in green tea possess CaP chemopreventive and possibly therapeutic effects. This present study was designed to compare CaP cancer chemopreventive effects of green tea polyphenols (GTP), water extract of black tea, and their major constituents epigallocatechin-3-gallate and theaflavins, respectively, in athymic nude mice implanted with androgen-sensitive human CaP CWR22Rn1 cells. Our data demonstrated that the treatment with all the tea ingredients resulted in (i) significant inhibition in growth of implanted prostate tumors, (ii) reduction in the level of serum prostate specific antigen, (iii) induction of apoptosis accompanied with upregulation in Bax and decrease in Bcl-2 proteins, and (iv) decrease in the levels of VEGF protein. Furthermore, we also found that GTP (0.01 or 0.05% w/v; given after establishment of CWR22Rn1 tumor) causes a significant regression of tumors suggesting therapeutic effects of GTP at human achievable concentrations. Introduction Cancer of the prostate gland (CaP) is the most common invasive malignancy and a major cause of cancer related deaths of males in the USA. CaP is considered an ideal candidate disease for chemoprevention because it is typically detected in elderly population with a relatively slower rate of progression, and even a modest delay achieved via chemopreventive agents, Abbreviations: CaP, Cancer of the prostate gland; EGCG, epigallocatechin3-gallate; GTP, green tea polyphenols; PARP, poly ADP-ribose polymerase; PSA, prostate specific antigen; TF, theaflavins. # could have a significant impact on the outcome of this disease. Emerging studies, in the recent past, from our laboratory and by others as well as the epidemiological observations and some clinical trials have suggested that green tea or its polyphenols possess chemopreventive and therapeutic potential against CaP [(1,2) and references therein]. Green tea as well as most of the other types of teas (except herbal teas) is obtained from the leaves of tea plant Camelia sinensis. Although black tea is the most popular tea in the world, green tea is the most well studied form of tea for its anticancer effects. Both green and black teas contain abundant amounts of polyphenolic antioxidants and it has been shown that theaflavins (TF) in black tea and catechins in green tea are equally effective antioxidants (3). Studies from our laboratory have shown that oral infusion of green tea polyphenols (GTP), at a human achievable dose, inhibits the development of CaP and its subsequent metastasis in a transgenic adenocarcinoma of mouse prostate (TRAMP) model (1). Recently, we showed that the IGF/IGFBP3 signaling plays a crucial role during GTP-mediated inhibition of CaP and its metastasis in TRAMP mice (4). Caporali et al. (5) demonstrated the involvement of clusterin, a protein involved in many different processes, including apoptosis and neoplastic transformation, during CaP chemoprevention by GTP in TRAMP model. Recently, Pezzato et al. (6) demonstrated that the major polyphenol in green tea, epigallocatechin-3-gallate (EGCG) inhibited prostate specific antigen (PSA) expression and activities in prostate carcinoma cells. All these studies suggest that green tea possesses chemopreventive and possibly chemotherapeutic effects against CaP via modulating multiple pathways. However, more detailed studies in (i) appropriate animal models, and (ii) appropriate high-risk human population are needed to establish the chemopreventive and therapeutic potential of tea polyphenols for CaP. Further, the molecular mechanisms of the effects of tea polyphenols also need to be thoroughly examined for improving upon the prevalent approaches for CaP management. The present study was designed to determine and compare the chemopreventive and therapeutic effect of tea polyphenols, namely GTP, black tea polyphenols (as water extract of black tea leaves BTE), and their major constituents EGCG and TF, respectively, against CaP in athymic nude mice. For this purpose, we employed CWR22Rn1 cells, which is an androgen-responsive human CaP cell line derived from human primary CaP which forms rapid and reproducible tumor growth in nude mice and secretes PSA in the blood stream of the host. PSA is a clinical diagnostic tumor marker for monitoring the presence and progression of CaP in humans. The data from this study demonstrated that GTP and BTE as well as their major constituents EGCG and TF, respectively, impart a significant chemopreventive/therapeutic response against the development of prostate tumors in nude mice, without any apparent toxic response. The Author 2005. Published by Oxford University Press. All rights reserved. For Permissions, please email: [email protected] 833 I.A.Siddiqui et al. Materials and methods Cell line and reagents The androgen-responsive human prostate carcinoma CWR22Rn1 cells were obtained from ATCC (Manassas, VA). The cells were grown in RPMI 1640 (Mediatech, Herndon, VA) with 10% fetal bovine serum (Hyclone, Logan, UT), 100 U/ml penicillin, and 100 mg/ml streptomycin at 37 C in a 95% air and 5% CO2 humified atmosphere. Cells grown as monolayer were harvested by brief incubation with 0.05% trypsin–EDTA solution (Invitrogen, Carlsbad, CA) and used for xenograft implantation in nude mice. A purified preparation of EGCG and TF (>98% pure) were a kind gift from Dr Yukihiko Hara of Mitsui Norin Co. Ltd. (Shizuoka, Japan). GTP (>95% enriched preparation) was obtained from Natural Resources & Products (Charlottesville, VA). Chromatographic analysis of this mixture showed that it contains four major polyphenolic constituents: EGCG (62%), epicatechin-3-gallate (24%), epigallocatechin (5%) and epicatechin (6%). Black tea leaves were obtained from local market and the water extract was prepared by brewing the tea leaves in autoclaved water. Animals Athymic (nu/nu) nude mice (male; 6–8 weeks old) were obtained from NxGen Biosciences (San Diego, CA) and housed in the University of Wisconsin, Medical School Animal Care Facility at standard conditions (in laminar airflow cabinets under pathogen-free conditions with 12 h light/12 h dark schedule) and fed with autoclaved Harlan Teklad sterilizable rodent diet (Madison, WI) ad libitum. Implantation of tumors in nude mice and treatments with tea polyphenols This study was performed to analyze the chemopreventive/therapeutic effects of tea polyphenols against CaP in athymic nude mice implanted with androgensensitive human prostate carcinoma CWR22Rn1 cells. The rationale for the choice of CWR22Rn1 cells is based on the fact that our major goal was to determine the chemopreventive effects of tea polyphenols in early stages of CaP development, when the disease is androgen-dependent. Another reason for using CWR22Rn1 cells was that they make PSA, which is arguably considered a ‘gold standard’ for monitoring the CaP in humans. The two different experimental protocols employed are described below. Protocol-1. Under this experimental protocol we studied and compared the CaP chemopreventive potential of GTP and BTE as well as their major constituents, EGCG and TF, respectively. In this experiment, we employed the CWR22Rn1 cells for implantation in athymic nude mice. In our experiment, 50 nude mice were randomly divided into five groups of 10 animals each. CWR22Rn1 cells (1 · 106 cells) were suspended in 1:1 medium mixed with Matrigel and were subcutaneously inoculated on the left and right flanks of each mouse. The details of treatments are given below. Group I: Control-cells were implanted (at the start of the experiment; Day 0), no further treatment was given; Group II: GTP-cells were implanted on Day 0 followed by providing freshly prepared solution of 0.1% GTP in autoclaved water (every Monday, Wednesday and Friday) ad libitum (starting at Day 1); Group III: BTE-cells were implanted on Day 0 followed by freshly prepared 1.25% black tea extract in autoclaved water (supplied every Monday, Wednesday and Friday) ad libitum (starting at Day 1); Group IV: EGCGcells were implanted on Day 0 followed by a daily treatment of EGCG [1 mg/day/mice in phosphate-buffered saline (PBS); pH 7.4, i.p.) starting at day 1; Group V: TF-cells were implanted on Day 0 followed by a daily treatment of TF (1 mg/day/mice in PBS; pH 7.4, i.p.) starting at Day 1. EGCG and TF were prepared fresh on each day and injected within 30 min of their preparation. The treatment schedule was continued until the tumors reached a volume of 1200 mm3. At this point, the animals were withdrawn from the study and euthanized. Throughout the experiment the animals were housed under standard housing conditions and had free access to autoclaved laboratory chow diet. Blood was withdrawn periodically to determine the effects of treatments on PSA levels in serum. In this protocol, to assess the possibility of treatment-toxicity, the effect of treatments on food consumption and body weight was monitored twice weekly and the fluid consumption was monitored every Monday, Wednesday and Friday throughout the study. Protocol-2. This protocol was designed to assess the therapeutic potential of GTP against CaP at physiologically achievable doses of the test material. For this purpose, CWR22Rn1 cells were inoculated onto the flanks of athymic nude mice as described under protocol 1. The animals were given autoclaved diet and water ad libitum until the tumors were established to a volume of 400 mm3. At this point, the animals were evenly divided into three groups and treatment was started. The details of treatment are as follows. 834 Group I: control-cells were implanted (at the start of the experiment; Day 0), no further treatment was given; Group II: cells were implanted and tumors were allowed to grow to a volume of 400 mm3, followed by oral infusion (ad libitum) with 0.05% solution of GTP in autoclaved water as the sole source of drinking fluid (supplied every Monday, Wednesday and Friday); Group III: cells were implanted and tumors were allowed to grow to a volume of 400 mm3, followed by oral infusion (ad libitum) with 0.01% solution of GTP in autoclaved water as the sole source of drinking fluid (supplied every Monday, Wednesday and Friday). The treatment was continued until the tumor reached to a volume of 1200 mm3. At this point, the animals were withdrawn from the study and euthanized. Tumor size and volume At different times post-cell implantation, the size of the tumor was measured by determining two perpendicular dimensions and the height using vernier caliper. The tumor volume was calculated using the formula V ¼ 1/2(4p/3) (L1/2) (L2/2) (H) ¼ 0.5238 L1L2H, where L1 is the long diameter, L2 is short diameter and H is the height (7). At the termination of these studies, tumors were excised, snap frozen in liquid nitrogen and kept at 80 C for further use. Collection of blood and determination of serum prostate specific antigen levels The effect of tea polyphenols on CaP development was also determined by following PSA levels in the serum. Blood was collected by ‘madibular bleed’ and serum was separated by allowing the blood to clot at room temperature for about an hour followed by centrifuging it for 10 min at 4 C. The levels of PSA were determined by using a quantitative human PSA enzyme-linked immunosorbent assay (ELISA) kit (Anogen, Ontario, Canada) as per the manufacturer’s protocol. For PSA determination, serum was used within 24–48 h of isolation or samples were stored at 80 C till further use. Immunoblot analysis For immunoblot analysis, a portion of tumor tissue was thawed on ice and homogenized in lysis buffer (50 mM Tris–HCl, 150 mM NaCl, 1 mM EGTA, 1 mM EDTA, 20 mM NaF, 100 mM Na3VO4, 0.5% NP40, 1% Triton X-100, 1 mM PMSF, 10 mg/ml aprotinin and 10 mg/ml leupeptin, pH 7.4) at 4 C to prepare tissue lysate. The protein concentration was determined using BCA protein assay kit (Pierce Biotechnology, Rockford, IL) as per the vendor’s instructions. Appropriate amount of protein (25–50 mg) was resolved over 8–14% Tris–glycine polyacrylamide gel and then transferred onto the nitrocellulose membrane. The blots were blocked using 5% non-fat dry milk and probed using appropriate primary antibody in blocking buffer overnight at 4 C. The membrane was then incubated with appropriate secondary antibody horseradish peroxidase conjugate (Amersham Life Sciences Inc., Arlington Heights, IL) followed by detection using chemiluminescence ECL kit (Amersham Life Sciences Inc.). The antibodies used for this purpose were anti-Bax, anti-Bcl-2, anti-poly ADP-ribose polymerase (PARP), (Upsate USA, Charlottesville, VA), anti-VEGF (Santa Cruz Biotechnology, Santa Cruz, CA) and anti-caspase 3 (Cell Signaling Technology, Beverly, MA). Equal loading of protein was confirmed by stripping the membrane and re-probing it with monoclonal bactin primary antibody (Sigma, St Louis, MO). Statistical analysis The time to a target mean tumor volume of 1200 mm3 is defined as the elapsed time from the date of cell implantation to the date when a 1200 mm3 target is reached or when the mouse was euthanized. A Kaplan–Meier survival analysis with the corresponding log-rank analysis was performed using S-plus Software (Insightful; Seattle, WA). A P-value of 0.05 was considered to be statistically significant. To assess the effect of oral feeding and intra-peritoneal injections of tea and tea polyphenols on protein expressions and PSA levels, comparisons were made between controls and tea treated groups using a 2-tailed Student’s t-test. Values of P < 0.05 were considered statistically significant. Results The major aim of this study was to determine and compare the chemopreventive/anti-proliferative effects of GTP and BTE as well as their major constituents EGCG and TF, respectively, against the development of CWR22Rn1 cell implanted tumors in nude mice. Therefore, as the first step in our approach, we studied and compared the CaP chemopreventive potential of GTP, BTE, and their major constituents EGCG and TF, respectively. In this protocol, to assess the effect of treatments on toxicity, the food and fluid consumption and body weight Inhibition of CWR22Rn1 tumor growth and PSA secretion were monitored throughout the study. Our data demonstrated that GTP and BTE given as sole source of drinking fluid and TF and EGCG when given as i.p. injections did not result in any apparent toxicity in terms of food and fluid consumption as well as body weight (data not shown). As shown in Figure 1, the treatment of mice with GTP, BTE, and their major constituents EGCG and TF, respectively, was found to result in a significant inhibition in growth of implanted tumors. In this protocol, we euthanized the animals when the tumor reached a volume of 1200 mm3. Thus, as shown in Figure 1, in control animals, the tumor volume of 1200 mm3 was reached in 26 days post-tumor cell inoculation. The most effective tumor growth inhibitory response was observed in GTP-treated group where the targeted tumor volume of 1200 mm3 was reached on day 54 post-tumor cell inoculation. Other treatments were also found to be significantly effective. The tumor volume of 1200 mm3 in animals in BTE group reached in 42 days, whereas it took 43 and 38 days; respectively, in the animals treated with TF and EGCG. The Kaplan–Meier plot is given in Figure 2A. The observed differences for GTP, BTE and TF mice as compared with untreated mice were statistically significant with P < 0.01 according to a log-rank analysis (Figure 2B). The observed differences for all treated mice as compared with untreated mice were statistically significant with P < 0.01. Our data demonstrated that in all the analyses, GTP treatment was most effective among the agents tested (P < 0.001). However, BTE, TF and EGCG treatments were also found to impart significant (P < 0.01) tumor growth inhibitory effects (Figure 2A and B). Next, we determined the effect of various tea polyphenols on serum PSA levels in nude mice implanted with CWR22Rn1 tumors. As shown by the data in Figure 3, at day 18 post-tumor inoculation, the level of PSA in control animals was found to be 13.95 ± 0.82 ng/ml; while in animals treated with GTP, BTE, EGCG and TF, the PSA levels were found to decrease significantly to 4.95 ± 1.23, 6.37 ± 1.75, 4.07 ± 0.98 and 5.9 ± 0.78 ng/ml, respectively (P < 0.01). Similarly at day 24 post-inoculation, the level of PSA in control animals was 26.4 ± 1.32 ng/ml; whereas in animals treated with GTP, BTE, EGCG and TF, the PSA levels were found to significantly decrease respectively to 4.46 ± 1.32, 8.85 ± 2.32, 5.7 ± 0.58 and 6.9 ± 1.32 ng/ml (P < 0.01) (Figure 3). PSA levels were also determined at earlier times; however, the values were found to be <4 ng/ml (considered negative as per the criterion of the kit), the data are not shown here. Thus, our data clearly demonstrated that GTP and BTE as well as their major constituents resulted in a significant decrease in the serum PSA levels in athymic nude mice implanted with human CaP cells. Our next aim was to determine the mechanisms of prostate tumor growth inhibition in nude mice. Because tea polyphenols have been shown to induce apoptosis of cancer cells, we determined the effect of tea polyphenols on PARP, caspase 3 and Bcl-2 family of proteins, all of which are known to regulate 1400 Tumor volume (mm3) 1200 1000 800 600 Control 400 GTP BTE EGCG 200 TF 0 20 24 28 32 36 40 44 48 52 Days post-treatment Fig. 1. Effect of GTP and BTE as well as their major polyphenols on the growth of CWR22Rn1 implanted tumors in athymic nude mice. CWR22Rn1 cells (1 · 106) were implanted on the right and left flanks of athymic nude mice and the mice were treated with GTP and BTE and their polyphenols as described in Materials and methods. The animals were followed for the development of tumors and the experiment terminated when the tumor volume reached 1200 mm3. Results are expressed as tumor volume (mm3) ± SD of 10 mice. Fig. 2. Statistical evaluation of the effect of GTP and BTE as well as their major polyphenols on the growth of CWR22Rn1 implanted tumors in athymic nude mice. CWR22Rn1 cells (1 · 106) were implanted on the right and left flanks of athymic nude mice and the mice were treated with GTP and BTE and their polyphenols as described in Materials and methods. The animals were followed for the development of tumors and the experiment terminated when the tumor volume reached 1200 mm3. (A) The tumor incidence was analyzed by Kaplan–Meier analysis to evaluate mice with tumor volume <1200 mm3. (B) Log-rank analysis of the Kaplan–Meier data to determine number of days for tumors to reach a volume of 1200 mm3. Results are expressed as tumor volume (mm3) ± SD of 10 mice. P < 0.001, P < 0.01 was considered to be statistically significant compared with the untreated controls. 835 I.A.Siddiqui et al. Fig. 3. Effect of GTP and BTE as well as their major polyphenols on the serum PSA levels of athymic nude implanted with CWR22Rn1 cells. CWR22Rn1 cells (1 · 106) were implanted on the right and left flanks of athymic nude mice and the mice were treated with GTP and BTE and their polyphenols. The mice were bled every week and the blood was collected by ‘madibular bleed’ and serum was separated as described in Materials and methods. The levels of PSA were determined by using a quantitative human PSA enzyme linked immunosorbent assay. Results are expressed as PSA levels (ng/ml serum) ± SD of 10 mice. P < 0.01 was considered to be statistically significant compared with the untreated controls. programmed cell death (8–10). As shown by the western blot analysis, all the tea polyphenols under investigation were found to induce an increase in active caspase 3 and cleaved PARP levels indicating an induction of apoptosis by tea polyphenols. Interestingly, tea polyphenols resulted in an appreciable decrease in the levels of pro-form of caspase 3 (Figure 4). We next studied the effect of tea polyphenols on Bax and Bcl-2 protein, which are the most important members of Bcl-2 family of proteins. As shown in Figure 4, tea polyphenols resulted in an upregulation in Bax and decrease in Bcl-2 proteins; thereby increasing the Bax/Bcl-2 ratio to favor induction of apoptosis. These results suggested that the tumor inhibitory effects of tea polyphenols are mediated via an increased apoptosis in tumors and this induction of apoptosis is caused via a modulation of Bcl-2 family of proteins. Because our recent study has shown that GTP causes an inhibition of angiogenesis in prostate (4), we next compared the effects of various tea polyphenols on VEGF protein levels in prostate tumors from nude mice. Interestingly, as shown in the Figure 4, the levels of VEGF protein were found to be significantly inhibited by all the preparations of tea polyphenols examined (Figure 4). Based on the outcome of this study, in the next protocol, we decided to perform an experiment to ascertain the therapeutic potential of GTP at a dose that could be physiologically achievable. For this purpose we decided to choose 0.05 and 0.01% of GTP given as sole source of drinking fluid. Our data demonstrated that GTP (0.05 and 0.01%) treatment resulted in an inhibition of CWR22Rn1 tumor growth in nude mice (Figure 5). Our data demonstrated that the 0.05% dose of GTP resulted in a significant tumor inhibition as compared with 0.01% dose (Figure 5). The Kaplan–Meier plot of the tumor data is given in Figure 5B. The time to reach an average tumor 836 Fig. 4. Effect of GTP and BTE as well as their major polyphenols on the protein expression of caspase 3, PARP, Bax, Bcl-2 and VEGF in CWR22Rn1 tumors. CWR22Rn1 cells (1 · 106) were implanted on the right and left flanks of athymic nude mice and the mice were treated with GTP and BTE and their polyphenols as described in Materials and methods. The animals were followed for the development of tumors and the experiment terminated when the tumor volume reached 1200 mm3 and the tumors were excised. Tissue lysates were subjected to immunoblot analysis for each specific protein. Equal loading of the proteins were confirmed by stripping the blots and re-probing with b-actin. Western blot analysis was conducted in five animals in each group, and only representative blots for two animals are shown. Lanes 1 and 2 ¼ Control; lanes 3 and 4 ¼ GTP (0.1% w/v) treated; lanes 5 and 6 ¼ BTE (1.25% w/v) treated; lanes 7 and 8 ¼ EGCG (1 mg/kg body wt) treated; lanes 9 and 10 ¼ TF (1 mg/kg body wt) treated. volume of 1200 mm3 was 39 days post-inoculation in the control mice, 44 days for 0.01% GTP mice and 51 days for 0.05% GTP mice (Figure 5B). Using log-rank analysis (Figure 5C) a significant difference in tumor growth was observed in 0.05% GTP mice as compared with the controls (P < 0.01). Thus, our data confirmed the observations in the previous experiment and demonstrated a significant delay in tumor formation with physiologically achievable concentrations of GTP. Discussion In vitro as well as in vivo studies from our laboratory and elsewhere have suggested that tea polyphenols (especially from green tea) could impart chemopreventive and possibly therapeutic effect against CaP (1,2,11,12), which is a major health problem in the United States and in other parts of the world (13,14). While most of the work has been done with GTP, only limited studies have evaluated black tea polyphenols for their CaP chemopreventive potential (15–17), and no study has assessed the comparative chemopreventive/ therapeutic effects of green and black tea polyphenols against CaP. Therefore, we designed this study to evaluate the Inhibition of CWR22Rn1 tumor growth and PSA secretion Fig. 5. Effect of GTP on the growth of CWR22Rn1 implanted tumors in athymic nude mice. CWR22Rn1 cells (1 · 106) were implanted on the right and left flanks of athymic nude mice and the mice were treated with two doses of GTP as described in Materials and methods (protocol 2). The animals were followed for the development of tumors and the experiment terminated when the tumor volume reached 1200 mm3. (A) Growth of tumors as a function of time. (B) Kaplan–Meier analysis of the tumor data. (C) Log-rank analysis of the Kaplan–Meier data. Results are expressed as tumor volume (mm3) ± SD of 10 mice. P < 0.01 was considered to be statistically significant as compared with untreated controls. comparative chemopreventive/therapeutics potential of different preparations of tea polyphenols against CaP in prostate tumor implants in athymic nude mice. In the first approach, we conducted a detailed study with GTP and BTE and their major polyphenolic constituents EGCG and TF, respectively. For this study, we used CWR22Rn1 cells because these cells are known to secrete PSA and PSA is considered the ‘gold standard’ for monitoring the CaP in humans. As discussed under Results, our data clearly demonstrated that all the preparation of tea polyphenols cause a significant inhibition in tumor growth and the serum PSA levels in athymic nude mice implanted with CWR22Rn1 cells. This is an important observation because serum PSA is considered to be an important marker for identifying humans for adenocarcinoma of the prostate (18,19). In the clinic, serum PSA measurements are being widely used to screen for CaP (20–23). Several investigators have reported the usefulness of serum PSA as a follow up marker for local recurrence and/or distant disease in the patients after radical prostatectomy, radiation and hormonal therapy (18,19). Despite of a continuing debate, at the present time, serum PSA is the regarded as the best marker for volume of CaP (tumor burden) in men (24–26). Our data also demonstrated that GTP, BTE or their major constituents EGCG and TF, at observed doses, did not cause any apparent toxicity (body weight and fluid consumption) in mice. Further, there was no observed change in consumption of food in various treatment groups. This is consistent with earlier studies where tea and its polyphenols did not show any apparent sign of toxicity in vivo (1,4,27–29). Next, we assessed the mechanisms involved in the CaP chemopreventive effects of tea polyphenols. Our data suggested that the growth inhibition of CWR22Rn1 tumor xenograft by tea polyphenols is associated with an increase in apoptosis of tumor cells. The induction of apoptosis was evident from our data showing the increase in active caspase 3 and cleaved PARP levels in the tumors. Caspase 3 is an intracellular cysteine protease that exists as a proenzyme, becoming activated during the sequence of events associated with apoptosis. Caspase 3 cleaves a variety of 837 I.A.Siddiqui et al. cellular molecules that contain the amino acid theme DEVD such as PARP, the 70 kD protein of the U1-ribonucleoprotein and a subunit of the DNA dependent protein kinase. Thus, our findings are consistent with in vitro studies where treatment of CaP cells with tea polyphenols resulted in apoptosis and cell cycle arrest (2,11,30–32). Further, our study demonstrated that increased apoptosis in the tumors is associated with down modulation of Bcl-2 and a concomitant increase in Bax. Bcl-2 is an anti-apoptotic gene and, the link between apoptosis and cancer emerged when Bcl-2 (B-cell lymphoma 2), which is the gene that is linked to an immunoglobulin locus by chromosome translocation in follicular lymphoma, was found to inhibit cell death (33). This unexpected discovery gave birth to the concept, now widely embraced that impaired apoptosis is a crucial step in the process of cancer development [(34,35) and the references therein]. In the present study, we have shown that tea polyphenols resulted in a significant decrease in the levels of anti-apoptotic Bcl-2 protein and increase in the pro-apoptotic Bax protein thus shifting the Bax/Bcl-2 ratio in favor of apoptosis. Studies have shown that Bcl-2 forms a heterodimer with Bax and might thereby neutralize its proapoptotic effects (36,37). In addition, Bcl-2 is also known to prevent the release of caspases (37–39). It is also important to mention here that our findings are consistent with other studies where a similar observation in cell culture was observed (8–10). Thus, our data suggested that the observed tumor inhibition by tea polyphenols may be associated with modulations in Bcl-2-family proteins that favor apoptosis of the tumor cells in vivo. We also determined the modulations in VEGF, a marker protein for angiogenesis, during the observed tumor inhibition by tea polyphenols. Angiogenesis is a physiological process of formation of new blood vessels and this process is always stimulated in tumors (40,41). Our data demonstrated that the protein level of VEGF was appreciably inhibited by tea polyphenols. The inhibition of VEGF by green tea has been shown previously in a model of corneal neovascularization (27). In this model, drinking green tea (1.25% in drinking water) was found to be associated with significant inhibition of VEGF-induced corneal neovascularization. Because the growth of all solid tumors is dependent on angiogenesis, inhibition of VEGF by green tea could explain why drinking green tea prevents the growth of a variety of tumors. Based on the outcome of our detailed study, we decided to assess the therapeutic potential of GTP against CaP at low concentration which could be easily achievable physiologically following green tea consumption. As apparent under Results, our data clearly verified that both the doses of GTP resulted in a significant inhibition in tumor growth when given after the tumors were established to a volume of 400 mm3. Taken together, our study suggested that all preparations of tea (black as well as green tea) were effective in inhibiting the growth of prostate tumors in athymic nude mice and in addition to the chemopreventive effects, GTP also has cancer therapeutic potential at physiologically achievable concentrations. Our data also suggested that the CaP chemopreventive effect of tea polyphenols are mediated via (i) induction of apoptosis of tumor cells that is caused by modulations in Bcl-2 family of proteins, which favor apoptosis, and (ii) inhibition of angiogenesis in tumors. The outcome from this study could thus have a direct practical implication and translational relevance to human CaP patients. 838 Acknowledgements This work was supported by US PHS Grants RO1 CA 78809, RO1 CA 101039 and P50 DK065303-01. Conflict of Interest Statement: None declared. References 1. Gupta,S., Hastak,K., Ahmad,N., Lewin,J.S. and Mukhtar,H. (2001) Inhibition of prostate carcinogenesis in TRAMP mice by oral infusion of green tea polyphenols. Proc. Natl. Acad. Sci. USA, 98, 10350–10355. 2. Ahmad,N., Feyes,D.K., Nieminen,A.L., Agarwal,R. and Mukhtar,H. (1997) Green tea constituent epigallocatechin-3-gallate and induction of apoptosis and cell cycle arrest in human carcinoma cells. J. Natl. 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