Cannabis Research A-Z

Sami Sarfaraz, Farrukh Afaq, Vaqar M. Adhami, et al.

Cancer Res 2005;65:1635-1641. Published online March 7, 2005.

 Updated Version

 Copyright © 2005 American Association for Cancer Research

 Sami Sarfaraz, Farrukh Afaq, Vaqar M. Adhami, and Hasan Mukhtar
Department of Dermatology, University of Wisconsin, Madison, Wisconsin
Abstract
Cannabinoids, the active components of Cannabis sativa
Linnaeus (marijuana) and their derivatives have received
renewed interest in recent years due to their diverse
pharmacologic activities such as cell growth inhibition,
anti-inflammatory effects and tumor regression. Here we
show that expression levels of both cannabinoid receptors,
CB1 and CB2, are significantly higher in CA-human papillomavirus-
10 (virally transformed cells derived from adenocarcinoma
of human prostate tissue), and other human
prostate cells LNCaP, DUI45, PC3, and CWR22RN1 than in
human prostate epithelial and PZ-HPV-7 (virally transformed
cells derived from normal human prostate tissue) cells. WIN-
55,212-2 (mixed CB1/CB2 agonist) treatment with androgenresponsive
LNCaP cells resulted in a dose- (1-10 Mmol/L) and
time-dependent (24-48 hours) inhibition of cell growth,
blocking of CB1 and CB2 receptors by their antagonists
SR141716 (CB1) and SR144528 (CB2) significantly prevented
this effect. Extending this observation, we found that WIN-
55,212-2 treatment with LNCaP resulted in a dose- (1-10
Mmol/L) and time-dependent (24-72 hours) induction of
apoptosis (a), decrease in protein and mRNA expression of
androgen receptor (b), decrease in intracellular protein and
mRNA expression of prostate-specific antigen (c), decrease in
secreted prostate-specific antigen levels (d), and decrease in
protein expression of proliferation cell nuclear antigen and
vascular endothelial growth factor (e). Our results suggest
that WIN-55,212-2 or other non–habit-forming cannabinoid
receptor agonists could be developed as novel therapeutic
agents for the treatment of prostate cancer. (Cancer Res 2005;
65(5): 1635-41)
Introduction
Because prostate cancer has become the most common cancer
diagnosed in men, developing novel targets and mechanism-based
agents for its treatment has become a challenging issue. In recent
years cannabinoids, the active components of Cannabis sativa
Linnaeus (marijuana) and their derivatives have drawn renewed
attention because of their diverse pharmacologic activities such
as cell growth inhibition, anti-inflammatory effects, and tumor
regression (1–5). Cannabinoids have been shown to induce
apoptosis in gliomas (6), PC-12 pheochromocytoma (7), CHP
100 neuroblastoma (8), and hippocampal neurons (9) in vitro, and
most interestingly, regression of C6-cell gliomas in vivo (10).
Further interest in cannabinoid research came from the discovery
of specific cannabinoid systems and the cloning of specific
cannabinoid receptors (10). These diversified effects of cannabinoids
are now known to be mediated by the activation of specific
G protein-coupled receptors that are normally bound by a family
of endogenous ligands, the endocannabinoids (11, 12). Two
different cannabinoid receptors have been characterized and
cloned from mammalian tissues: the ‘‘central’’ CB1 receptor (13),
and the ‘‘peripheral’’ CB2 receptor (14).
In the present study, we show for the first time that expression
levels of both cannabinoid receptors, CB1 and CB2, are higher in
human prostate cancer cells than in normal cells. Importantly, we
also show that WIN-55,212-2 (CB1/CB2 agonist) treatment with
androgen-responsive LNCaP cells results in a dose- and timedependent
inhibition of cell growth with a concomitant induction
of apoptosis, decrease in protein and mRNA expression of
androgen receptor and prostate-specific antigen (PSA), decrease
in secreted PSA levels, protein expression of proliferating cell
nuclear antigen (PCNA), and vascular endothelial growth factor
(VEGF). We suggest that cannabinoid receptor agonists may be
useful in the treatment of human prostate cancer.
Materials and Methods
Materials. R-(+)-WIN-55,212-2 (2,3 dihydro-5-methyl-3 [(morpholinyl)-
methyl]pyrollo (1,2,3 de)-1,4-benzoxazinyl]-[1-naphthaleny]methanone,
C27H26N2O3
CH3SO3H was purchased from Sigma (St. Louis, MO). CB1
receptor antagonist SR141716 (SR1) and CB2 receptor antagonist SR144528
(SR2) were procured from Dr. Herbert H. Seltzman (National Institute on
Drug Abuse, Division of Neuroscience and Behavioral Research, through RTI
International, Research Triangle Park, NC). DMEM and fetal bovine serum
were procured from Life Technologies, Invitrogen Corporation (Grand
Island, NY). Human PSA ELISA kit from Yes Biotech Laboratories (Ontario,
Canada) and annexin-V-FLUOS staining kit were from Roche Diagnostic
Corporation (Indianapolis, IN). Antibiotics (penicillin and streptomycin)
used were obtained from Cellgro Mediatech, Inc. (Herndon, VA). Apo-Direct
kit for measuring apoptosis by flow cytometry was procured from Apo-
Direct (San Diego, CA). RNA isolation kit was from Qiagen, Inc. (Valencia,
CA). Monoclonal antibodies for PSA, androgen receptor, PCNA, and VEGF,
were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Antimouse
secondary horseradish peroxidase conjugate was obtained from
Amersham Biosciences Limited (Buckinghamshire, United Kingdom).
Protein was estimated using bicinchoninic acid protein assay kit obtained
from Pierce (Rockford, IL).
Cell Culture. The LNCaP, DU145, PC-3, CWR22Rr1, CA-HPV-10, and
PZ-HPV-7 cells were obtained from American Type Culture Collection
(Manassas, VA). LNCaP and DU145 cells were cultured in DMEM
supplemented with 5% heat-inactivated fetal bovine serum and 1%
antibiotic penicillin and streptomycin. PC-3 and CWR22Rr1 cells were
cultured in RPMI 1640 medium supplemented with 5% heat-inactivated fetal
bovine serum and 1% antibiotic penicillin and streptomycin. CA-HPV-10
and PZ-HPV-7 were grown in keratinocyte serum-free medium (17005-042,
Life Technologies) supplemented with 5 ng/mL human recombinant EGF
and 50 Ag/mL bovine pituitary extract. Human prostate epithelial cells
(PrEC) were obtained from Cambrex Bioscience (Walkersville, MD) and
grown in prostate epithelial basal cell medium (Cambrex Bioscience)
Requests for reprints: Hasan Mukhtar, Department of Dermatology, University of
Wisconsin, Medical Sciences Center, Room B-25, 1300 University Avenue, Madison, WI
53706. Phone: 608-263-3927; Fax: 608-263-5223; E-mail: hmukhtar@wisc.edu.
I2005 American Association for Cancer Research.
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according to the manufacturer’s instructions. The cells were maintained
under standard cell culture conditions at 37jC and 5% CO2 in a humid
environment.
Treatment of Cells. WIN-55,212-2 (dissolved in DMSO), was used for
the treatment of cells. The final concentration of DMSO used was 0.1%
(v/v) for each treatment. For dose-dependent studies, cells were treated
with WIN-55,212-2 at final concentrations of 1.0, 2.5, 5.0, 7.5, and 10.0
Amol/L for 24 hours in complete cell medium, whereas for timedependent
studies, the cells (50-60% confluent) were treated with 5
Amol/L dose of WIN-55,212-2 for 24, 48, and 72 hours. For timedependent
study, we included a control treated with DMSO for 72 hours
because it was the longest time point post-WIN-55,212-2 treatment in our
experimental protocol. To establish the role of CB1 and CB2 receptors in
WIN-55,212-2–induced inhibitory effects, two experiments were done. In
the first experiment, cells were treated with 2 Amol/L of SR141716 or
SR144528 alone for 24 hours. In the second experiment, cells pretreated
with each of these antagonists (2 Amol/L) for 3 hours followed by
incubation with 7.5 Amol/L WIN-55,212-2 for 24 hours. In pilot
experiments, it was established that DMSO (0.1% v/v) had no effects
when measured at 24, 48, or 72 hours.
Cell Viability. The effect of WIN-55,212-2 on the viability of cells was
determined by (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazoliumbromide)
(MTT) assay. The cells were plated at 1  104 cells per well in 200
AL of complete culture medium containing 1.0, 2.5, 5.0, 7.5, and 10.0
Amol/L concentrations of WIN-55,212-2 in 96-well microtiter plates for 24
and 48 hours at 37jC in a humidified chamber. Each concentration of
WIN-55,212-2 was repeated in 10 wells. After incubation for specified
times at 37jC in a humidified incubator, MTT reagent (4 AL, 5 mg/mL in
PBS) was added to each well and incubated for 2 hours. The microtiter
plate containing the cells was centrifuged at 1,800 rpm for 5 minutes at
4jC. The MTT solution was removed from the wells by aspiration and
the formazan crystals were dissolved in DMSO (150 AL). Absorbance was
recorded on a microplate reader at 540 nm wavelength. The effect of
WIN-55,212-2 on growth inhibition was assessed as the percentage of
inhibition in cell growth where vehicle-treated cells were taken as
100% viable.
Preparation of Cell Lysates and Western Blot Analysis. Following
treatment of cells with WIN-55,212-2, the medium was aspirated and the
cells were washed with cold PBS [10 mmol/L (pH 7.45)]. The cells were
then incubated in ice-cold lysis buffer (50 mmol/L Tris-HCl, 150 mmol/L
NaCl, 1 mmol/L EGTA, 1 mmol/L EDTA, 20 mmol/L NaF, 100 mmol/L
Na3VO4, 0.5% NP40, 1% Triton X-100, 1 mmol/L phenylmethylsulfonyl
fluoride (pH 7.4)], with freshly added protease inhibitor cocktail (protease
inhibitor cocktail set III, Calbiochem, La Jolla, CA) over ice for 20
minutes. The cells were scraped and the lysate was collected in a
microfuge tube and passed through a 21.5-gauge needle to break up the
cell aggregates. The lysate was cleared by centrifugation at 14,000  g
for 15 minutes at 4jC, and the supernatant (total cell lysate) was
collected, aliquoted, and used on the day of preparation or immediately
stored at 80jC for use at a later time. For Western blotting, 25 to 50
Ag of protein were resolved over 12% polyacrylamide gels and transferred
onto a nitrocellulose membrane. The nonspecific sites on blots were
blocked by incubating in blocking buffer [5% nonfat dry milk/1% Tween
20 in 20 mmol/L TBS (pH 7.6)] for 1 hour at room temperature,
incubated with appropriate monoclonal primary antibody in blocking
buffer for 90 minutes to overnight at 4jC, followed by incubation with
anti-mouse or anti-rabbit secondary antibody horseradish peroxidase
conjugate and detected by chemiluminescence and autoradiography
using Hyperfilm obtained from Amersham Biosciences. Densitometric
measurements of the bands in Western blot analysis were done using
digitalized scientific software program UN-SCAN-IT purchased from Silk
Scientific Corporation (Orem, UT).
ELISA for PSA. The human PSA ELISA kit was used for the
quantitative determination of PSA levels in culture medium according
to the vendor’s protocol. This kit uses a technique of quantitative
sandwich immunoassay for determination of PSA with an estimated
sensitivity of 1 ng/mL PSA antigen.
Detection of Apoptosis and Necrosis by Confocal Microscopy. The
annexin-V-FLUOS staining kit was used for the detection of apoptotic and
necrotic cells according to vendor’s protocol. This kit uses a dual-staining
protocol in which the apoptotic cells are stained with annexin-V (green
fluorescence), and the necrotic cells are stained with propidium iodide (PI;
red fluorescence). LNCaP cells were grown to about 70% confluence and
then treated with WIN-55,212-2 (1.0, 2.5, 5.0, 7.5, and 10.0 Amol/L) for 24
hours. The fluorescence was measured by a Zeiss 410 confocal microscope
(Thornwood, NY). Confocal images of green annexin-FITC fluorescence
were collected using 488 nm excitation light from an argon/krypton laser,
a 560 nm dichroic mirror, and a 514 to 540 nm bandpass barrier filter.
Images of red PI fluorescence were collected using a 568 nm excitation
light from the argon/krypton laser, a 560 nm dichroic mirror, and a 590-
nm-long pass filter. In a selected field, the cells stained with annexin-V
and PI as well as unstained cells were counted to ascertain the extent of
apoptosis and necrosis.
Quantification of Apoptosis by Flow Cytometry. The cells were grown
at density of 1  106 cells in 100 mm culture dishes and were treated with
WIN-55,212-2 (1.0, 2.5, 5.0, 7.5, and 10.0 Amol/L doses) for 24 hours. The
cells were trypsinized, washed with PBS, and processed for labeling with
fluorescein-tagged dUTP nucleotide and PI by use of an Apo-Direct
apoptosis kit obtained from Phoenix Flow Systems (San Diego, CA) and
was used according to the manufacturer’s protocol. The labeled cells were
analyzed by flow cytometry.
Quantitative Real-time PCR for mRNA Expression of Androgen
Receptor and PSA. Total RNA was isolated from LNCaP cells using RNeasy
kit according to the vendor’s protocol. The ratio of optical densities of RNA
samples at 260 and 280 nm was consistently >1.8. cDNA was synthesized by
reverse transcription of 1 Ag of extracted RNA with 200 units of Moloney
murine leukemia virus reverse transcriptase (Promega, Madison, WI) in the
presence of oligo dT and deoxynucleotide triphosphate (Promega).
Androgen receptor and PSA were amplified using a Failsafe real-time
PCR system obtained from Epicentre (Madison, WI). The thermal cycler
used for amplification was an ABI-PRISM 7000 sequence detection
system (Applied Biosystems, Foster City, CA). Primers were designed
as follows: androgen receptor forward, 5V-AAGACGCTTCTACCAGCTCACCAA;
reverse, 5V-TCCCAGAAAGGATCTTGGGCACTT; PSA forward,
5V-ACTCACAGCAAGGATGGAGCTGAA; reverse, 5V-TGAGGGTTGTCTGGAGGACTTCAA.
The cycler was programmed with the following
conditions (a) initial denaturation at 94jC for 2 minutes, followed by
35 cycles of (b) 94jC for 40 seconds, (c) annealing of the primer template
at 58jC for 40 seconds, and (d) extension at 72jC for 40 seconds.
Results
Expression of Cannabinoid Receptor in Normal and
Prostate Cancer Cells. We first compared the expression levels
of both cannabinoid receptors CB1 and CB2 in PrEC and a series of
human prostate cancer cells. We also included a pair of cells, PZHPV-
7 (virally transformed cells, derived from normal human
prostate tissue) and CA-HPV-10 (virally transformed cells, derived
from the adenocarcinoma of human prostate tissue) derived from
the same individual. Immunoblot data shown in Fig. 1 revealed that
expression of both CB1 and CB2 receptors was significantly higher
in prostate cancer cells LNCaP, DUI45, PC3, CWR22Rr1, and CAHPV-
10 as compared with normal prostate cells PZ-HPV-7 and
PrEC cells. To establish the specificity of the cannabinoid receptor
antibodies used in the blotting experiments, antigen preabsorption
experiments were carried out. The peptides blocked anti-CB1 and
anti-CB2 antibody binding in all cells (data not shown).
Effect of WIN-55,212-2 on Cell Viability of PrEC and LNCaP
Cells. To evaluate the cell viability response of WIN-55,212-2 on
PrEC and LNCaP cells, MTT assay was employed. Data in Fig. 2A
shows that treatment of PrEC cells with WIN-55,212-2 (1-10 Amol/L)
for 24 and 48 hours had no effect on cell viability (Fig. 2A). However,
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treatment of LNCaP cells with similar doses of WIN-55,212-2 in a
dose-dependent manner significantly decreased the viability of
LNCaP cells at 24 and 48 hours (Fig. 2A). The IC50 for inhibition of
cell viability at 24 and 48 hours was 6.0 and 5.0 Amol/L, respectively.
CB1 and CB2 receptor activation signals growth inhibition in
LNCaP cells. To study the possible implication of CB1 and CB2
receptors in WIN-55,212-2–induced cell death, the effect of their
antagonists were evaluated using MTT assay. Cells pretreated with
2 Amol/L of SR141716 (CB1 antagonist) or SR144528 (CB2
antagonist) had no effect on cell viability but exhibited significant
protective effect when coadministered with WIN-55,212-2 (7.5
Amol/L) at a molar ratio of 1:3.75 (Fig. 2B). These data suggest
that both CB1 and CB2 receptors may be involved in WIN-55,212-
2–mediated growth inhibition and apoptosis.
Effect of WIN-55,212-2 on Apoptosis and Necrosis of LNCaP
Cells. We next assessed whether the cell growth inhibitory effect
of WIN-55,212-2 was associated with induction of apoptosis. The
induction of apoptosis by WIN-55,212-2 was evident from the
analysis of the data obtained by confocal microscopy after
labeling the cells with annexin-V (Fig. 2B). This method was used
because it identifies the apoptotic (green fluorescence) as well as
necrotic (red fluorescence) cells. As shown by the data, WIN-
55,212-2 treatment resulted in a dose-dependent apoptosis in
LNCaP cells. In a time-dependent study with a 5 Amol/L dose of
WIN-55,212-2, there was an increasing trend of apoptotic cells at
72 compared with 48 hours after treatment (Fig. 2C). We next
quantified the extent of apoptosis by flow cytometric analysis of
the cells labeled with dUTP and PI. LNCaP cells were treated
with of WIN-55,212-2 (1-10 Amol/L) for 24 hours. As shown by
the data in Fig. 2D, WIN-55,212-2 treatment resulted in 18.3%
and 25.6% of apoptotic cells at a dose of 7.5 and 10 Amol/L,
respectively. Whereas the induction of apoptosis was almost
negligible at the lowest dose (1.0 Amol/L) used, the highest dose
employed (10 Amol/L) resulted in a massive induction of
apoptosis as determined by flow cytometry. A similar trend
was evident when apoptosis was measured by ladder formation
on agarose gel electrophoresis (data not shown).
Effect of WIN-55,212-2 on Androgen Receptor and PSA
Protein and mRNA Expression in LNCaP Cells. Androgens are
involved in the development and progression of prostate cancer
where androgen receptor is assumed to be the essential mediator
for androgen action (15, 16). In the next series of experiments, we
determined the effect of WIN-55,212-2 on protein and mRNA
expression of androgen receptor. In a dose-dependent study, we
found that treatment of LNCaP cells with WIN-55,212-2 resulted
in a marked decrease in androgen receptor protein expression
(Fig. 3A). Relative density data of these immunoblots revealed
that the decrease in androgen receptor protein expression was
50% and 90% at 5.0 and 7.5 Amol/L of WIN-55,212-2, respectively.
In a time-dependent study with 5 Amol/L dose of WIN-55,212-2,
there was a marked decrease in androgen receptor protein
expression and this corresponded with the relative density data
showing a decrease of 61% and 69% at 48 and 72 hours,
respectively (Fig. 3B). Studies have also shown that modulation in
androgen receptor leads to alteration in androgen-responsive
genes (17). PSA is an androgen-responsive gene and is regarded as
the most sensitive biomarker and screening tool for prostate
cancer in humans (18). The dose-dependent effect of WIN-55,212-
2 on LNCaP cells showed a significant decrease in PSA protein
expression at 5.0, 7.5, and 10 Amol/L concentrations when
assessing at 24 hours post-treatment (Fig. 3A). Densitometric
analysis data revealed that the decrease was 48%, 75%, and 90% at
5.0, 7.5, and 10.0 Amol/L concentrations (Fig. 3A). For timedependent
studies, cells were treated with 5 Amol/L of WIN-
55,212-2 for 24, 48, and 72 hours. Employing Western blot
analysis, we found a significant decrease in a time-dependent
manner in PSA protein expression. Densitometric analysis
revealed a decrease in PSA protein expression by 48% and 60%
at 48 and 72 hours, respectively (Fig. 3B). We also evaluated the
effect of WIN-55,212-2 on mRNA levels of androgen receptor and
PSA. As shown by the real time-PCR analysis data, there was an
inhibition in mRNA levels of androgen receptor (Fig. 3C) and PSA
(Fig. 3D) at 7.5 and 10.0 Amol/L concentrations.
We next examined the effect of WIN-55,212-2 on secreted
levels of PSA in LNCaP cells. Employing ELISA technique, we
found that treatment of LNCaP cells with WIN-55,212-2 resulted
in a dose-dependent decrease in the secreted levels of PSA by
30%, 53%, and 62% at 5.0, 7.5, and 10 Amol/L, respectively. At
similar doses of WIN-55,212-2, but varying the time point by 48
hours, PSA levels decreased by 53%, 77%, and 80% (Fig. 3E).
Furthermore, at 72 hours post-treatment of WIN-55,212-2,
secreted PSA levels decreased by 58%, 82%, and 88% (Fig. 3E).
From these data, it seems that the decrease in LNCaP cell
growth was concomitant with a decrease in androgen receptor
protein expression as well as a decrease in both intracellular and
secreted PSA levels.
Effect of WIN-55,212-2 on Cell Proliferation Marker, PCNA.
We next determined the effect of WIN-55,212-2 on PCNA which
serves as a requisite auxiliary protein for DNA polymerase y-driven
DNA synthesis and is cell-regulated (19, 20). The dose-dependent
study treatment of LNCaP cells with WIN-55,212-2 (1-10 Amol/L)
resulted in a significant decrease in protein expression of PCNA.
Western blot analysis and relative density of these bands showed
that the decrease in protein expression of PCNA was 71% at 7.5
Amol/L WIN-55,212-2 (Fig. 4A). In a time-dependent study,
treatment of LNCaP cells with 5 Amol/L WIN-55,212-2 resulted in
>50% inhibition in PCNA protein expression at 48 and 72 hours of
treatment (Fig. 4B).
Figure 1. Western blot analysis of CB1 and CB2
cannabinoid receptor expression in normal and
human prostate cancer cells. A, a pair of normal
(PZ-HPV-7) and prostate cancer cells
(CA-HPV-10) obtained from the same individual;
B, PrEC (normal prostate epithelial cells) and
prostate cancer cells, LNCaP, DU145, PC-3,
and CWR22Rr1. Total cell lysates were prepared
and 30 Ag of protein were subjected to
SDS-PAGE, followed by immunoblot analysis
and chemiluminescence detection. h-Actin was
used as a loading control. For other details, see
Materials and Methods.
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Effect of WIN-55,212-2 on VEGF. Because VEGF is a marker
for angiogenesis, blocking the angiogenic process may represent
a promising way of treating the tumor. Studies have shown that
androgen regulates VEGF content in prostate cancer (21). As
WIN-55,212-2 treatment resulted in a decrease in androgen
receptor expression, the effects on VEGF were also determined.
It was observed that WIN-55,212-2 treatment also resulted in a
decrease in VEGF protein expression (Fig. 4A). Densitometric
analysis data showed a decrease of 40% at 7.5 Amol/L
concentration of WIN-55,212-2. In a time-dependent study at
5 Amol/L WIN-55,212-2 treatment, VEGF protein expression
decreased in a time-dependent manner (Fig. 4B).
Discussion
It is now well accepted that uncontrolled cellular growth,
which may be a result of defects in cell cycle and apoptotic
machinery, is responsible for the development of most of the
cancers including prostate cancer. Thus, the agents which can
modulate apoptosis in cancer cells may be able to affect the
Figure 2. Effect of WIN-55,212-2 on cell viability and apoptosis in LNCaP cells. A, effects on the viability of PrEC and LNCaP cells. As detailed in Materials and
Methods, the cells were treated with WIN-55,212-2 (1-10 Amol/L) for 24 and 48 hours, and their viability was determined by MTT assay. Columns, means; bars, F SE
of three separate experiments in which each treatment was done in 10 wells; *, P < 0.001 compared with control (0 Amol/L). B, effects of CB1 receptor antagonist
SR141716 and CB2 receptor antagonist SR144528 on WIN-55,212-2–induced cell viability. As detailed in Materials and Methods, cells were treated with 2 Amol/L
of SR141716 or SR144528 alone for 24 hours. In another parallel set, cells were pretreated with each of these antagonists for 3 hours, followed by incubation with
7.5 Amol/L WIN-55,212-2 for 24 hours and their viability was determined by MTT assay. Columns, means; bars, F SE of three separate experiments in which each
treatment was done in 10 wells; *, P < 0.001 compared with WIN. C and D, induction of apoptosis by confocal microscopy, cells were treated with WIN-55,212-2
(1-10 Amol/L) for 24 hours for the dose-dependent study (C), and WIN-55,212-2 (5 Amol/L) for 24, 48, and 72 hours for the time-dependent study (D). The
annexin-V-FLUOS staining kit was used for the detection of apoptotic and necrotic cells. This kit uses a dual-staining protocol in which apoptotic cells are stained
with annexin-V (green fluorescence) and necrotic cells are stained with PI (red fluorescence). E, quantification of apoptosis by flow cytometry. The cells were
treated with WIN-55, 212-2 (1-10 Amol/L) for 24 hours, labeled with dUTP using terminal deoxynucleotide transferase and PI. Cells showing fluorescence (R2) are
considered as apoptotic cells and their percentage population is indicated. Data from representative experiments repeated thrice with similar results.
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steady-state cell population and may be useful in the management
and therapy of cancer. Consistent with this notion, there is
a need to develop novel targets and mechanism-based agents for
the management of prostate cancer. One of the most exciting and
promising areas of current cannabinoid research is the ability of
these compounds to control the cell survival/death decision (1).
In this study, we found that compared with PrEC and PZ-HPV-7
cells, the expression levels of both cannabinoid receptors CB1 and
CB2 were significantly higher in CA-HPV-10 and other human
prostate cells LNCaP, DU145, PC-3, and CWR22Rr1. These data
suggest that CB1 and CB2 receptors could be a target for novel
treatment options for prostate cancer. We also found that mixed
CB1/CB2 agonist WIN-55,212-2 treatment of LNCaP cells resulted
in a decrease of cell viability as determined by MTT assay at
varying doses and time points (Fig. 2A), suggesting the
involvement of both CB1 and CB2 in the antiproliferative action
of cannabinoids (Fig. 2B). It is widely recognized that apoptosis is
an ideal way of elimination of cancer cells and that selective
apoptotic events could provide suitable targets for cancer
treatment and prevention. In this study, we also observed an
increase in apoptosis of LNCaP cells by treatment with WIN-
55,212-2. This observation was confirmed by employing confocal
microscopy (Fig. 2C and D) and flow cytometry (Fig. 2E). This
could be an important observation which might be useful for
devising strategies for the management of human prostate cancer
because apoptosis is a physiological and discrete way of cell death
different from necrotic cell death and is regarded to be an ideal
way of cell elimination.
Androgens are essential for the growth, differentiation, and
functioning of the prostate as well as in increasing prostate cancer
development (22, 23). Many molecular mechanisms have been
suggested for the development of recurrent hormone refractory
Figure 3. Effect of WIN-55,212-2 on protein and mRNA expression of androgen receptor and PSA in LNCaP cells. A, dose-dependent effect; and B, time-dependent
effect. As detailed in Materials and Methods, the cells were treated with DMSO alone or with specified concentrations of WIN-55,212-2 in DMSO and then harvested.
Total cell lysates were prepared and 30 Ag of protein were subjected to SDS-PAGE, followed by immunoblot analysis and chemiluminescence detection. The
values above the blots represent change as compared with vehicle treatment in protein expression of the bands normalized to h-actin. Western blot data from a
representative experiment repeated thrice with similar results. C and D, effects of WIN-55,212-2 on mRNA expression of androgen receptor (C) and PSA (D) determined
by real time-PCR from representative experiments repeated twice with similar results. E, effect on secreted levels of PSA. Cells were treated with WIN-55,212-2
(1-10 Amol/L) for 24, 48, and 72 hours and then harvested. The PSA levels were determined by ELISA as described under Materials and Methods. Points, means; bars,
F SE of three independent experiments.
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tumors. Most of these mechanisms postulate an alteration in the
function of the androgen receptor and its signaling pathways (23).
The overexpression of androgen receptor in prostate cancer may
promote cell growth. Hence, elimination or reducing the androgen
receptor in prostate cancer should help in treating this neoplastic
disease. As most of the molecular mechanism for the development of
prostate cancer involves modulation in the function of androgen
receptor and its signaling pathways, we further studied the effect of
WIN-55,212-2 on androgen receptor protein and mRNA expression
and its subsequent effect on PSA production. Our results indicate
that WIN-55,212-2 treatment significantly decreases androgen
receptor protein (Fig. 3A) and mRNA expression (Fig. 3C) in
LNCaP cells.
PSA belongs to the kallikrein family (17), is a serine protease with
highly prostate-specific expression, and is the most widely employed
marker in the detection of early prostate cancer. For these reasons, it
is considered that agents which could reduce PSA levels may have
important clinical implications for prostate cancer. Earlier studies
reported that PSA is primarily regulated by androgens (24). This
observation was based on the fact that the antiandrogen,
cyperoterone acetate, had the ability to induce PSA, and that
hydroxyflutamide could block androgen and progesterone induction
of PSA glycoprotein, thus suggesting that PSA glycoprotein
expression is influenced predominantly by androgens via its
receptor, and the mutation of the receptor can affect the expression
of this gene by steroids other than androgens (24). Recent studies
have established that androgen receptor functions as a transcriptional
regulator via its binding to androgen response elements
within promoter and enhancer regions of PSA. PSA is currently the
most accepted marker for assessment of prostate cancer progression
in humans and is being detected in the serum of patients with
prostate diseases including prostatitis, benign prostatic hypertrophy,
and prostate cancer (18). It is reported that in LNCaP cells,
androgens regulate PSA glycoprotein expression and mRNA via
androgen receptor (25, 26). Our studies show a significant decrease
in intracellular, mRNA (Fig. 3D), as well as secreted levels of PSA by
WIN-55,212-2 treatment of cells, suggesting that cannabinoid
receptor agonists may be exploited to prevent prostate cancer
progression.
PCNA recognizes nuclear antigens and its overexpression is
associated with increase in PSA serum levels (27). PCNA
expression has significant prognostic value and it seems to be a
significant biomarker in prognosis and treatment of prostate
cancer (27). Our results also suggest that concomitant with the
decrease in PCNA protein expression (Fig. 4A), there was a
decrease in PSA serum levels following WIN-55,212-2 treatment
(Fig. 3E).
VEGF is a ubiquitous cytokine that regulates embryonic
vasculogenesis and angiogenesis. Normal prostate epithelium
expresses low levels of VEGF, whereas premalignant lesions have
increased VEGF expression, which is additionally increased in
prostate carcinoma (28). Studies have shown that cannabinoid
treatment markedly reduced the expression of VEGF in gliomas,
the most potent proangiogenic factor and also of angiopoietin 2,
which contributes to the angiogenic process by preventing vessel
maturation (29). Our results showed that treatment of LNCaP
with WIN-55,212-2 inhibits growth and VEGF protein expression
(Fig. 4A and B).
Recently, cannabinoids have received considerable attention due
to their diverse pharmacologic activities such as cell growth
inhibition, anti-inflammatory effects, and tumor regression. Our
results suggest that treatment of androgen-responsive human
prostate carcinoma LNCaP cells resulted in a decrease in
intracellular and secreted levels of PSA, with concomitant
inhibition of androgen receptor, cell growth, and induction of
apoptosis. We conclude that cannabinoids should be considered as
agents for the management of prostate cancer. If our hypothesis is
supported by in vivo experiments, then the long-term implications
of our work could be to develop non–habit-forming cannabinoid
agonist(s) for the management of prostate cancer.
Figure 4. Effect of WIN-55,212-2 on protein expression of
PCNA and VEGF in LNCaP cells. A, dose-dependent effect;
B, time-dependent effect. As detailed in Materials and Methods,
the cells were treated with DMSO alone or specified
concentrations of WIN-55,212-2 in DMSO and then harvested.
Total cell lysates were prepared and 30 Ag of proteins were
subjected to SDS-PAGE, followed by immunoblot analysis and
chemiluminescence detection. The values above the blots
represent change as compared with vehicle treatment in protein
expression of the bands normalized to h-actin. Data from a
representative experiment repeated thrice with similar results.
Cancer Research
Cancer Res 2005; 65: (5). March 1, 2005 1640 www.aacrjournals.org
Copyright © 2005 American Association for Cancer Research
Downloaded from cancerres.aacrjournals.org on April 11, 2011
DOI:10.1158/0008-5472.CAN-04-3410
Acknowledgments
Received 9/20/2004; revised 12/16/2004; accepted 1/4/2005.
Grant support: Department of Defence idea development award W81XWH-04-
1-0217.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
We thank Dr. Nihal Ahmad for helpful discussions and critical reading of the
manuscript.
References
1. Guzman M. Cannabinoids: potential anticancer agents.
Nat Rev Cancer 2003;3:745–55.
2. Casanova ML, Blazquez C, Martinez-Palacio J, et al.
Inhibition of skin tumor growth and angiogenesis
in vivo by activation of cannabinoid receptors. J Clin
Invest 2003;111:43–50.
3. Galve-Roperh I, Sanchez C, CortesML,Gomezdel Pulgar
T, Izquierdo M, Guzman M. Anti-tumoral action of
cannabinoids: involvement of sustained ceramide accumulation
and extracellular signal-regulated kinase activation.
Nat Med 2000;6:313–9.
4. Sanchez C, de Ceballos ML, del Pulgar TG, et al.
Inhibition of glioma growth in vivo by selective
activation of the CB(2) cannabinoid receptor. Cancer
Res 2001;61:5784–9.
5. Bifulco M, Laezza C, Portella G, et al. Control by the
endogenous cannabinoid system of ras oncogenedependent
tumor growth. FASEB J 2001;15:2745–7.
6. Velasco G, Galve-Roperh I, Sanchez C, Blazquez C,
Guzman M. Hypothesis: cannabinoid therapy for the
treatment of gliomas. Neuropharmacology 2004;47:
315–23.
7. Sarker KP, Obara S, Nakata M, Kitajima I, Maruyama I.
Anandamide induces apoptosis of PC-12 cells: involvement
of superoxide and caspase-3. FEBS Lett 2000;
472:6:1039–44.
8. Maccarrone M, Lorenzon T, Bari M, Melino G,
Finazzi-Agro A. Anandamide induces apoptosis in
human cells via vanilloid receptors. Evidence for a
protective role of cannabinoid receptors. J Biol Chem
2000;275:31938–45.
9. Chan GC, Hinds TR, Impey S, Storm DR. Hippocampal
neurotoxicity of D9-tetrahydrocannabinol. J Neurosci
1998;18:5322–32.
10. Guzman M, Sanchez C, Galve-Roperh I. Control of
the cell survival/death decision by cannabinoids. J Mol
Med 2001;78:613–25.
11. Devane WA, Hanus L, Breuer A, et al. Isolation and
structure of a brain constituent that binds to the
cannabinoid receptor. Science 1992;258:1946–9.
12. Porter AC, Felder CC. The endocannabinoid nervous
system: unique opportunities for therapeutic intervention.
Pharmacol Ther 2001;90:45–60.
13. Matsuda LA, Lolait SJ, Brownstein MJ, Young AC,
Bonner TI. Structure of a cannabinoid receptor and
functional expression of the cloned cDNA. Nature 1990;
346:561–4.
14. Munro S, Thomas KL, Abu-Shaar M. Molecular
characterization of a peripheral receptor for cannabinoids.
Nature 1993;365:61–5.
15. Lamb DJ, Weigel NL, Marcelli M. Androgen receptors
and their biology. Vitam Horm 2001;62:199–230.
16. Wang LG, Liu XM, Kreis W, Budman DR. Downregulation
of prostate-specific antigen expression by
finasteride through inhibition of complex formation
between androgen receptor and steroid receptorbinding
consensus in the promoter of the PSA gene in
LNCaP cells. Cancer Res 1997;57:714–9.
17. Naz RK, Herness EA. Prostate-specific genes:
present status and future direction. Front Biosci
2001;6:1083–8.
18. Stamey TA, Yang N, Hay AR, McNeal JE, Freiha FS,
Redwine E. Prostate-specific antigen as a serum marker
for adenocarcinoma of the prostate. N Engl J Med
1987;317:909–16.
19. Prosperi E. Multiple roles of the proliferating cell
nuclear antigen: DNA replication, repair and cell cycle
control. Prog Cell Cycle Res 1997;3:193–210.
20. Kelman Z. PCNA: structure, functions and interactions.
Oncogene 1997;14:629–40.
21. Joseph IB, Nelson JB, Denmeade SR, Isaacs JT.
Androgens regulate vascular endothelial growth factor
content in normal and malignant prostatic tissue. Clin
Cancer Res 1997;12:2507–11.
22. Lindzey J, Kumar MV, Grossmann M, Young CYF,
Tindall J. Molecular mechanisms of androgen action.
Vitam Horm 1994;49:383–432.
23. Koivisto P, Kolmer M, Visakorpi T, Kallioniemi OP.
Androgen receptor gene and hormonal therapy failure
of prostate cancer. Am J Pathol 1998;152:1–9.
24. Montgomery BT, Young CY, Bilhartz DT, et al.
Hormonal regulation of prostate-specific antigen
(PSA) glycoprotein in the human prostatic adenocarcinoma
cell line, LNCaP. Prostate 1992;21:63–73.
25. Lee C, Sutkowski DM, Sensibar JA, et al. Regulation
of proliferation and production of prostate-specific
antigen in androgen-sensitive prostatic cancer cells,
LNCaP, by dihydrotestosterone. Endocrinology 1995;
136:796–803.
26. Henttu P, Lukkarinen O, Vihko P. Expression of the
gene coding for human prostate-specific antigen and
related hGK-1 in benign and malignant tumors of the
human prostate. Int J Cancer 1990;45:654–60.
27. Bantis A, Giannopoulos A, Gonidi M, et al.
Expression of p120, Ki-67 and PCNA as proliferation
biomarkers in imprint smears of prostate carcinoma
and their prognostic value. Cytopathology 2004;1:
25–31.
28. Mazzucchelli R, Montironi R, Santinelli A, Lucarini G,
Pugnaloni A, Biagini G. Vascular endothelial growth
factor expression and capillary architecture in high
grade PIN and prostate cancer in untreated and
androgen-ablated patients. Prostate 2000;45:72–9.
29. Blazquez C, Casanova ML, Planas A, et al. Inhibition
of tumor angiogenesis by cannabinoids. FASEB J
2003;17:529–31.

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Revised 16 July 2009; Accepted 21 July 2009; Published online 18 August 2009

British Journal of Cancer (2009) www.bjcancer.com

 N Olea-Herrero,  D Vara, S Malagarie-Cazenave, Díaz-Laviada

Correspondence: Professor I Díaz-Laviada, E-mail: ines.diazlaviada@uah.es

Abstract

Carolyn Churchill

19 Aug 2009

Chemicals found in cannabis could help treat prostate cancer, according to a new study.

Researchers discovered that two cannabinoids, the active chemicals found in the drug, prevent cancer cells from multiplying and in some instances actually kill the cells.

But cancer experts warned people suffering from prostate cancer not to treat themselves by smoking cannabis as more work is needed to explore ways in which the chemicals could be used to benefit patients.

Dr Lesley Walker, Cancer Research UK’s director of cancer information, said: “This is interesting research which opens a new avenue to explore potential drug targets but it is at a very early stage – it absolutely isn’t the case that men might be able to fight prostate cancer by smoking cannabis.

 This is interesting research that opens up a new avenue to explore Dr Lesley Walker, Cancer Research UK’s director of cancer information

“This research suggest that prostate cancer cells might stop growing if they are treated with chemicals found in cannabis but more work needs to be done to explore the potential of the cannabinoids in treatment.”

Prostate cancer is the second most common form of the disease among men in Scotland and about 2500 new cases are diagnosed every year. A total of 793 men died from prostate cancer in 2007 and official figures show that 19.9% of patients with the disease will have died within five years of diagnosis.

Researchers from the University of Alcala in Madrid used artificial molecules that are similar to two cannabinoids and tested their effect on prostate cancer cells grown in a lab. They also carried out tests on mice that had been transplanted with human prostate cells.

The researchers found that if the cannabinoids bind to a receptor called CB2, the cancer cells stop multiplying and in some instances die. When the CB2 receptor was switched off, the prostate cancer cells carried on dividing and growing.

Cancer Research UK said the study, published in the British Journal of Cancer, suggested that the CB2 receptor, one of two forms of cannabinoid receptors, plays a pivotal role in stopping the spread of prostate cancer cells.

The other receptor, CB1, is responsible for the psychoactive effects of cannabis so it is hoped that the new discovery will allow scientists to concentrate on the development of drugs targeting the CB2 receptor, which would enable prostate cancer to be treated without causing the same side-effects.

Professor Ines Diaz-­Laviada, study author at the University of Alcala, said: “Our research shows that there are areas on prostate cancer cells which can recognise and talk to chemicals found in cannabis called cannabinoids.

“These chemicals can stop the division and growth of prostate cancer cells and could become a target for new research into potential drugs to treat prostate cancer.”

The potential medicinal benefits of cannabis, which is a Class B drug, were recognised in the nineteenth century and Queen Victoria was rumoured to use the drug to treat menstrual pains.

Since then, research has been ongoing to discover new ways to use the drug to treat a range of conditions.

2003 Jun 15

Mimeault M, Pommery N, Wattez N, Bailly C, Hénichart JP.

Institut de Chimie Pharmaceutique Albert Lespagnol, 3 Rue du Professeur Laguesse, BP83, Lille, France.

Abstract

BACKGROUND: Anandamide (ANA) is an endogenous lipid which acts as a cannabinoid receptor ligand and with potent anticarcinogenic activity in several cancer cell types.

METHODS: The inhibitory effect of ANA on the epidermal growth factor receptor (EGFR) levels expressed on the EGF-stimulated prostatic cancer cells LNCaP, DU145, and PC3 was estimated by ELISA tests. The anti-proliferative and cytotoxic effects of ANA were also evaluated on these human prostatic cancer cell lines by growth tests, flow cytometric analyses, trypan blue dye exclusion assays combined with the Papanicolaou cytological staining method.

RESULTS: ANA induced a decrease of EGFR levels on LNCaP, DU145, and PC3 prostatic cancer cells by acting through cannabinoid CB(1) receptor subtype and this leaded to an inhibition of the EGF-stimulated growth of these cells. Moreover, the G(1) arrest of metastatic DU145 and PC3 growth was accompanied by a massive cell death by apoptosis and/or necrosis while LNCaP cells were less sensitive to cytotoxic effects of ANA. The apoptotic/necrotic responses induced by ANA on these prostatic cancer cells were also potentiated by the acidic ceramidase inhibitor, N-oleoylethanolamine and partially inhibited by the specific ceramide synthetase inhibitor, fumonisin B1 indicating that these cytotoxic actions of ANA might be induced via the cellular ceramide production.

CONCLUSIONS: The potent anti-proliferative and cytotoxic effects of ANA on metastatic prostatic cancer cells might provide basis for the design of new therapeutic agents for effective treatment of recurrent and invasive prostatic cancers.

Copyright 2003 Wiley-Liss, Inc.

Lidia Ruiz, Alberto Miguel, Inés Dı́az-Laviada

Received 30 July 1999

Endocrinology Vol. 141, No. 1 118-126
Copyright © 2000 by The Endocrine Society

Endocrinology, doi:10.1210/en.141.1.118

  Dominique Melck, Luciano De Petrocellis, Pierangelo Orlando, Tiziana Bisogno, Chiara Laezza, Maurizio Bifulco and Vincenzo Di Marzo

Istituto per la Chimica di Molecole di Interesse Biologico (D.M., T.B., V.D.M.), Istituto di Cibernetica (L.D.P.), and Istituto di Biochimica delle Proteine ed Enzimologia (P.O.), Consiglio Nazionale delle Ricerche, 80072 Arco Felice (NA); and Centro di Endocrinologia e Oncologia Sperimentale, Consiglio Nazionale delle Ricerche, and Dipartimento di Biologia e Patologia Cellulare e Molecolare, Università di Napoli Federico II (C.L., M.B.), 80131 Naples, Italy

Address all correspondence and requests for reprints to: Dr. Vincenzo Di Marzo, Istituto per la Chimica di Molecole di Interesse Biologico, Consiglio Nazionale delle Ricerche, 80072 Arco Felice (NA), Italy. E-mail: vdm@trinc.icmib.na.cnr.it.

Anandamide and 2-arachidonoylglycerol (2-AG), two endogenous ligands of the CB1 and CB2 cannabinoid receptor subtypes, inhibit the proliferation of PRL-responsive human breast cancer cells (HBCCs) through down-regulation of the long form of the PRL receptor (PRLr). Here we report that 1) anandamide and 2-AG inhibit the nerve growth factor (NGF)-induced proliferation of HBCCs through suppression of the levels of NGF Trk receptors; 2) inhibition of PRLr levels results in inhibition of the proliferation of other PRL-responsive cells, the prostate cancer DU-145 cell line; and 3) CB1-like cannabinoid receptors are expressed in HBCCs and DU-145 cells and mediate the inhibition of cell proliferation and Trk/PRLr expression.

ß-NGF-induced HBCC proliferation was potently inhibited (IC₅₀ = 50–600 nM) by the synthetic cannabinoid HU-210, 2-AG, anandamide, and its metabolically stable analogs, but not by the anandamide congener, palmitoylethanolamide, or the selective agonist of CB2 cannabinoid receptors, BML-190. The effect of anandamide was blocked by the CB1 receptor antagonist, SR141716A, but not by the CB2 receptor antagonist, SR144528.

Anandamide and HU-210 exerted a strong inhibition of the levels of NGF Trk receptors as detected by Western immunoblotting; this effect was reversed by SR141716A. When induced by exogenous PRL, the proliferation of prostate DU-145 cells was potently inhibited (IC₅₀ = 100–300 nM) by anandamide, 2-AG, and HU-210. Anandamide also down-regulated the levels of PRLr in DU-145 cells. SR141716A attenuated these two effects of anandamide. HBCCs and DU-145 cells were shown to contain 1) transcripts for CB1 and, to a lesser extent, CB2 cannabinoid receptors, 2) specific binding sites for [³H]SR141716A that could be displaced by anandamide, and 3) a CB1 receptor-immunoreactive protein.

These findings suggest that endogenous cannabinoids and CB1 receptor agonists are potential negative effectors of PRL- and NGF-induced biological responses, at least in some cancer cells.