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 Raisuddin
1and Hasan Mukhtar
Department of Dermatology, University of Wisconsin-Madison, WI 53706, USA and1Department 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: hmukhtar@wisc.edu
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
invas-ive 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,
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
signal-ing plays a crucial role dursignal-ing 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
che-mopreventive 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.
Abbreviations:CaP, Cancer of the prostate gland; EGCG, epigallocatechin-3-gallate; GTP, green tea polyphenols; PARP, poly ADP-ribose polymerase; PSA, prostate specific antigen; TF, theaflavins.
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 100mg/ml streptomycin at 37C in a 95% air and 5% CO2humified 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 androgen-sensitive 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 con-stituents, 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: EGCG-cells 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.
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 V¼ 1/2(4p/3) (L1/2) (L2/2) (H)¼ 0.5238 L1L2H, where L1is the long diameter, L2is short diameter and H is the height (7). At the termination of these studies, tumors were excised, snap frozen in liquid nitrogen and kept at80C 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 4C. 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 at80C 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, 10mg/ml aprotinin and 10 mg/ml leupeptin, pH 7.4) at 4C 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 (25–50mg) was resolved over 8–14% Tris–glycine polyacrylamide gel and then transferred onto the nitro-cellulose membrane. The blots were blocked using 5% non-fat dry milk and probed using appropriate primary antibody in blocking buffer overnight at 4C. The membrane was then incubated with appropriate secondary antibody horse-radish 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 b-actin primary antibody (Sigma, St Louis, MO).
Statistical analysis
The time to a target mean tumor volume of 1200 mm3is defined as the elapsed time from the date of cell implantation to the date when a 1200 mm3target 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 of0.05 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
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 mm
3. Thus, as
shown in Figure 1, in control animals, the tumor volume of
1200 mm
3was reached in
26 days post-tumor cell
inocula-tion. The most effective tumor growth inhibitory response was
observed in GTP-treated group where the targeted tumor
vol-ume of 1200 mm
3was reached on day 54 post-tumor cell
inoculation. Other treatments were also found to be
signific-antly effective. The tumor volume of 1200 mm
3in 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
dif-ferences 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
signific-antly 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
polyphen-ols 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
Tumor volume (mm 3) 0 200 400 600 800 1000 1200 1400 20 24 28 32 36 40 44 48 52 Control GTP BTE EGCG TF 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.
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
poly-phenols. Interestingly, tea polyphenols resulted in an
appre-ciable 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
pro-teins; 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
apop-tosis in tumors and this induction of apopapop-tosis 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
polyphen-ols 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
achiev-able. 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
volume of 1200 mm
3was 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
concentra-tions 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
polyphen-ols 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
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. 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 mm3and 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 withb-actin. Western blot analysis was conducted in five animals in each group, and only
representative blots for two animals are shown. Lanes 1 and 2¼ Control; lanes 3 and 4¼ GTP (0.1% w/v) treated; lanes 5 and 6 ¼ BTE (1.25% w/v) treated; lanes 7 and 8¼ EGCG (1 mg/kg body wt) treated; lanes 9 and 10¼ TF (1 mg/kg body wt) treated.
comparative chemopreventive/therapeutics potential of
differ-ent 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
useful-ness of serum PSA as a follow up marker for local recurrence
and/or distant disease in the patients after radical
prostatec-tomy, 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
appar-ent 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
sugges-ted 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
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.
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
translo-cation in follicular lymphoma, was found to inhibit cell
death (33). This unexpected discovery gave birth to the
con-cept, 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
pro-apoptotic 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
physiologic-ally 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 mm
3.
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
thera-peutic 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.
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.
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Received September 12, 2005; revised October 30, 2005; accepted December 17, 2005.
