text:
CANCER - PROSTATE & Cannabis studies completed
Prostate cancer is a global public health problem, and it is the most common cancer in American men and the second cause for cancer-related death. Experimental evidence shows that prostate tissue possesses cannabinoid receptors and their stimulation results in anti-androgenic effects.
Prostate cancer starts in the cells of the prostate. The prostate is part of a man's reproductive system. Its main function is to make part of the liquid (seminal fluid) that mixes with sperm from the testicles to make semen. Semen is ejaculated during sex. The prostate is a walnut-sized gland just below the bladder and in front of the rectum. It surrounds part of the urethra, the tube that carries urine and semen through the penis.Are You 'Marijuana Deficient?'
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Cannabinoid Receptor as a Novel Target for the Treatment of Prostate Cancer
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
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)
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,
C27H26N2O3CH3SO3H 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: email@example.com.
I2005 American Association for Cancer Research.
www.aacrjournals.org 1635 Cancer Res 2005; 65: (5). March 1, 2005
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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
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.
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,
Cancer Research
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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.
Cannabinoid Receptor and Prostate Cancer
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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).
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.
Cancer Research
Cancer Res 2005; 65: (5). March 1, 2005 1638 www.aacrjournals.org
Copyright © 2005 American Association for Cancer Research
Downloaded from cancerres.aacrjournals.org on April 11, 2011
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.
Cannabinoid Receptor and Prostate Cancer
www.aacrjournals.org 1639 Cancer Res 2005; 65: (5). March 1, 2005
Copyright © 2005 American Association for Cancer Research
Downloaded from cancerres.aacrjournals.org on April 11, 2011
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
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
Received 9/20/2004; revised 12/16/2004; accepted 1/4/2005.
Grant support: Department of Defence idea development award W81XWH-04-
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
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Cannabis chemicals may help fight prostate cancer
LONDON | Wed Aug 19, 2009 2:22am EDT
(Reuters) - Chemicals in cannabis have been found to stop prostate cancer cells from growing in the laboratory, suggesting that cannabis-based medicines could one day help fight the disease, scientists said Wednesday.
After working initially with human cancer cell lines, Ines Diaz-Laviada and colleagues from the University of Alcala in Madrid also tested one compound on mice and discovered it produced a significant reduction in tumor growth.
Their research, published in the British Journal of Cancer, underlines the growing interest in the medical use of active chemicals called cannabinoids, which are found in marijuana.
Experts, however, stressed that the research was still exploratory and many more years of testing would be needed to work out how to apply the findings to the treatment of cancer in humans.
"This is interesting research which opens a new avenue to explore potential drug targets but it is at a very early stage," said Lesley Walker, director of cancer information at Cancer Research UK, which owns the journal.
"It absolutely isn't the case that men might be able to fight prostate cancer by smoking cannabis," she added
The cannabinoids tested by the Spanish team are thought to work against prostate cancer because they block a receptor, or molecular doorway, on the surface of tumour cells. This stops them from dividing.
In effect, the cancer cell receptors can recognize and "talk to" chemicals found in cannabis, said Diaz-Laviada.
"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," she said.
Her team's work with two cannabinoids -- called methanandamide and JWH-015 -- is the first demonstration that such cannabis chemicals prevent cancer cells from multiplying.
Some drug companies are already exploring the possibilities of cannabinoids in cancer, including British-based cannabis medicine specialist GW Pharmaceuticals.
It is collaborating with Japan's Otsuka on early-stage research into using cannabis extracts to tackle prostate cancer -- the most commonly diagnosed cancer in men -- as well as breast and brain cancer.
GW has already developed an under-the-tongue spray called Sativex for the relief of some of the symptoms of multiple sclerosis, which it plans to market in Europe with Bayer and Almirall.
Other attempts to exploit the cannibinoid system have met with mixed success. Sanofi-Aventis was forced to withdraw its weight-loss drug Acomplia from the market last year because of links to mental disorders.
(Editing by Simon Jessop)
Inhibition of human tumour prostate PC-3 cell growth by cannabinoids R(+)-Methanandamide and JWH-015: Involvement of CB2 - Abstract
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: email@example.com
We have previously shown that cannabinoids induce growth inhibition and apoptosis in prostate cancer PC-3 cells, which express high levels of cannabinoid receptor types 1 and 2 (CB1 and CB2). In this study, we investigated the role of CB2 receptor in the anti-proliferative action of cannabinoids and the signal transduction triggered by receptor ligation.
The human prostate cancer cell lines, namely PC-3, DU-145 and LNCaP, were used for this study. Cell proliferation was measured using MTT proliferation assay, [3H]-thymidine incorporation assay and cell-cycle study by flow cytometry. Ceramide quantification was performed using the DAG kinase method. The CB2 receptor was silenced with specific small interfering RNA, and was blocked pharmacologically with SR 144528. In vivo studies were conducted by the induction of prostate xenograft tumours in nude mice.
We found that the anandamide analogue, R(+)-Methanandamide (MET), as well as JWH-015, a synthetic CB2 agonist, exerted anti-proliferative effects in PC-3 cells. R(+)-Methanandamide- and JWH-015-induced cell death was rescued by treatment with the CB2 receptor antagonist, SR 144528. Downregulation of CB2 expression reversed the effects of JWH-015, confirming the involvement of CB2 in the pro-apoptotic effect of cannabinoids. Further analysing the mechanism of JWH-015-induced cell growth inhibition, we found that JWH-015 triggered a de novo synthesis of ceramide, which was involved in cannabinoid-induced cell death, insofar as blocking ceramide synthesis with Fumonisin B1 reduced cell death. Signalling pathways activated by JWH-015 included JNK (c-Jun N-terminal kinase) activation and Akt inhibition. In vivo treatment with JWH-015 caused a significant reduction in tumour growth in mice.
Chemicals found in cannabis may help fight prostate cancer
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.
Anti-proliferative and apoptotic effects of anandamide in human prostatic cancer cell lines: implication of epidermal growth factor receptor down-regulation and ceramide production
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.
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.
Δ9-Tetrahydrocannabinol induces apoptosis in human prostate PC-3 cells via a receptor-independent mechanism
Lidia Ruiz, Alberto Miguel, Inés Dı́az-Laviada
Received 30 July 1999
The effect of Δ9-tetrahydrocannabinol (THC), the major psycho-active component of marijuana, in human prostate cancer cells PC-3 was investigated. THC caused apoptosis in a dose-dependent manner. Morphological and biochemical changes induced by THC in prostate PC-3 cells shared the characteristics of an apoptotic phenomenon.
First, loss of plasma membrane asymmetry determined by fluorescent anexin V binding. Second, presence of apoptotic bodies and nuclear fragmentation observed by DNA staining with 4′,6-diamino-2-phenylindole (DAPI). Third, presence of typical 'ladder-patterned' DNA fragmentation. Central cannabinoid receptor expression was observed in PC-3 cells by immunofluorescence studies.
However, several results indicated that the apoptotic effect was cannabinoid receptor-independent, such as lack of an effect of the potent cannabinoid agonist WIN 55,212-2, inability of cannabinoid antagonist AM 251 to prevent cellular death caused by THC and absence of an effect of pertussis toxin pre-treatment.
Cancer Prevention & Survival Books
Suppression of Nerve Growth Factor Trk Receptors and Prolactin Receptors by Endocannabinoids Leads to Inhibition of Human Breast and Prostate Cancer Cell Proliferation
Endocrinology Vol. 141, No. 1 118-126
Copyright © 2000 by The Endocrine Society
Endocrinology, doi:10.1210/en.141.1.118
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: email@example.com.
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.


text:
Barcelona midfielder Arda Turan is set for an unspecified spell on the sidelines after returning from international duty with a hamstring injury. The La Liga giants have confirmed the blow ahead of a busy week which will see Ernesto Valverde's side hosting Espanyol on Saturday (9 September) three days before hosting Juventus in their first game of the Champions League group stage.
Turan, 30, made the Turkey's starting line-up in the 1-0 victory over Croatian before being replaced in the 70<sup>th minute of the game by new Celta Vigo signing Emre Mor.
It looks like the substitution was not tactical with Barcelona reporting that the midfielder returned from his international duties with a left hamstring strain.
The news shouldn't be a major concern for Valerde following a summer with plenty of speculation linking the former Atletico Madrid star with a move away from the Nou Camp.
Turan appears to be out of the manager's plans for the new season after failing to feature in any of the first four games of the 2017-2018 campaign – even being left out of the squad entirely during the defeats to Real Madrid in the Spanish Super Cup and in the 2-0 victory over Alaves.
Meanwhile, Ivan Rakitic could be ready to face Espanyol after being pictured alongside the rest of his available teammates during the Thursday's [7 September] training session.
Rakitic suffered an injury to his left thigh during Croatia's 1-0 victory over Kosovo, being consecutively ruled out for the World Cup qualifier against Turkey. However, the former Sevilla midfielder returned to training on Thursday and looks in line to be available for the upcoming derby against Espanyol and the Champions League visit of Juventus.
Meanwhile, Barcelona have confirmed that Lionel Messi, Javier Mascherano and the club's Spanish contingent were back in training after completing their international duties.
"Coach Ernesto Valverde is gradually getting his men back together at the Ciutat Esportiva. On Thursday another eight players returned to training for the morning session; Gerard Piqué, Jordi Alba, Sergio, Andrés Iniesta and Gerard Deulofeu after World Cup qualifiers with Spain, Leo Messi and Javier Mascherano after with Argentina and Arda Turan with Turkey," Barcelona confirmed in the club official website.
"The latter trained aside from the rest of the squad doing some specific work as he has a thigh strain and his recovery will determine his return. The majority of the squad continued their preparations for Saturday's Catalan derby against Espanyol at Camp Nou (8.45pm CET kick off). The squad will train again on Friday at 6.00pm CET in their last session before their third league game of the 2017/18 campaign."
Luis Suarez is yet to return but the Uruguay international is also expected to be ready for the visit of Espanyol after he played 90 minutes with 'no pain' during his national team's 2-1 victory over Paraguay this week.

subset: commoncrawl


text:
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Welcome to 45
I love celebrating my birthday. For me, it's always been a fun time. I'm not a big party kind of person. The task of organizing and getting people together doesn't really interest me. I'm more of a throw something together, tell me where to go, and I'll show up kind of person. I would rather throw a dinner party or have my friends meet at a restaurant.
I'm enjoy celebrating another year of life. Another year to live happily and the way I want to live. I am proud of the way I have lived my life so far. I have taken the time to figure out what I want to do, and I go do it. For the most part, I have done everything without a lot of struggle. My guiding principle for most decisions is: if it makes me happy and doesn't hurt anyone, I do it.
This year(and, for some reason, every odd year since 33), I freaked out a bit because I am turning 45. Where did the time go? Who I am and what I want has changed so much that I am just anxious for it to all fall into place. Then, I realized the phase that I just began will probably be the best time of my life. So what was is there to freak out about? Now, I am just as excited about my birthday as I have ever been.
This realization has caused me to reflect a bit. What have I learned so far? I came up with the 10 best lessons I learned in my first 45 years:
1. What it really means to love myself.
"You yourself, as much as anybody in the entire universe, deserve your love and affection." - Buddha
Loving yourself and being confident are not the same. Recognizing your good qualities is one thing, but really owning your self-worth is another. When you really love yourself, you don't put yourself last. You don't put yourself down. You don't allow someone else to put you down. You know you deserve whatever you want. You don't look for happiness outside of you because you know you're enough.
2. When to hold on and when to let go.
I am tenacious. I don't give up easily. I believe I am capable of doing anything that I have the desire to do. Yet, I have learned some situations are not going to be what I want them to be, and I had to let go. On the other end, you can hold on so tightly, you suffocate what it is that you want. When you just let go, things have a way of working out. It's all about trusting that whatever you want will show up for you the way it's supposed to.
3. Not everything is that serious.
Sometimes, you want something so badly that if it's not turning out the way you expect it to, you lose it. When did losing your shit ever make something better? If you
really think about it, most things aren't life threatening. The sun will rise, and you will too.
4. Speak the truth from my heart.
How I feel matters. As long as I speak from the heart and not the head. I will be okay.
5. I matter in the equation.
"Stop acting so small. You are the universe in ecstatic motion." - Rumi
I am always very aware of other people and how they feel. I am naturally empathetic, so I can see things from another's point of view. That's actually a gift but when that is a means of talking yourself out of what you want and putting your needs aside, then it's a problem.
6. If you focus on what you're afraid of, you get more of what you're afraid of.
"What we think, we become." - Buddha
I was in a friend's wedding, I wasn't a bridesmaid, but I knew three of them well. One of them was just a natural pessimist. Always afraid that something would go wrong. And in this wedding, one thing after another went wrong for her. There was a problem with her dress. She was the only bridesmaid who had a problem with her dress. The bride's mother offered to pay for the bridal party to get their hair done, She turned that down to have her own hairdresser do it. He still messed up her hair! She kept focusing on what could go wrong, and that's what she got. I've found when I focus on what's right with a situation, that's what shows up.
7. Be patient.
When I rush through something, I find I fuck it up more. So, now I just let it flow. I'm still working on this, but I am getting there.
8. Be present
I like to be where I am, I don't like to look back, I don't like to look too far ahead. Being present helps me get the most out of a situation.
9. If you can't control it, don't worry about it.
If I didn't organize something or have something to do with it to start with, I can't worry about how it will turn out. I can only focus on my part and make the best out
10. Don't give a fuck about what people think.
"Pay no mind to what they say… It doesn't matter anyway…" - The Go-Go's
I grew up with: what will they think? They were society, family, or a random person on the street. In my experience, most people aren't even paying that much attention to what someone else is doing. They're too wrapped up in their own thing. Even if you are gossiped about, people will bore of it quickly and gossip about the next thing. As long as I am happy with what I have done, I can't be wrapped up in what other people think.
This is just a small glimpse of what I have learned so far. I look forward to what life has in store for the next 45 years.
Until next time... look behind and beyond the veil...
Photo by Jim Strasma on Unsplash
Sacred Union
Divine feminine and divine masculine are apparent and required in all of creation. For example, electricity requires a plug and an outlet in order to access the electrical charge necessary to operate appliances, electric tools, or electronics. A vehicle requires gas in order to run which takes the phallus from the gas pump and the entrance into the gas tank to bring in the fuel that generates the energies for the car to run. In order to co-create a new life, the phallus of a man enters into the vagina of a woman and within this space new life is created.
The interesting part of the human make up is that as humans we have the spaciousness for the embodiment of both masculine & feminine characteristics and traits. In this article, I'm not suggesting physical traits, necessarily but more so within the way in which we operate. Once an awareness is brought into the attributes of both the divine feminine/masculine energies you are able to better understand your life and how to balance between the two in a way that actually serves you.
Characteristics of the Divine Masculine and Divine Feminine
What exactly do these traits look like? Below is a list of what makes up each side:
Divine Masculine
Fire energy
Make a plan
Execute goals
Stay in your own lane
Strong boundaries
Giver/ Producer
Guide yourself & others through fears
Reasoning mind
Masculine is direct- 'here is the deal'
Tangible outcomes & results
It's a thinking state
Some of the ways it can be experienced in an overactive way would be:
Only my way
Black and white thinking
Divine Feminine:
Water energy
Tapped into self & others
Able to Connect to other people on a soul level
Spacious - like the womb
It's a feeling state
Overactive or underactive:
Bleed into others
Not sure where you begin and others end
Lack of boundaries
In constant flow and can't actualize goals
Financial suffering
Recognizing imbalance
Recently, I worked with a female client who had been single for many years. She had hit a point in her life where she began to feel stuck. She was sensing a void, becoming hopeless, and feeling depressed. She expressed being tired of the daunting task of dating men who didn't take the initiative to ask her out on second dates and would leave all the planning up to her, Also, she was frazzled by the emotional state of the men she dated who she described as being sulky, pouty, unavailable and extremely passive.
She was reaching her mid- forties and feeling as if meeting a man who she could settle into a relationship and eventually have children with was out of her reach. The very thought of living life alone for the rest of forever was devastating to her. As we continued to communicate, she revealed that she created a life for herself by being driven for success within her career. Her lifestyle demanded rigid structure, schedules, and dedication. She was constantly in an action state of striving, working and achieving.
We also discovered that this lifestyle, even though it had worked well for her in the past was not working so well for her at the moment. At that point, she desired being connected to a mate and her feminine side. She wanted a family to nurture and wanted to be desired and supported herself.
Imbalance one way
In order for her to align with a mate that embodied more dominant divine masculine tendencies, it was important to shed light onto the ways in which her life was dominated by masculine tendencies, awaken her into her divine feminine attributes, and open the ability to be in a receptive state of being.
Because she operated within the dominant state of somewhat overactive masculine energies while being disconnected from her empowered divine feminine energies, it would be virtually impossible for her to attract a mate that operated within his divine masculine side. Hence, that is why the men she had dated predominantly embodied traits of the more feminine energies in a disempowered way.
Over a few short months of coaching, she was able to reconnect with her divine feminine side and shortly thereafter met her divine mate. She married him, and approximately 6 months later, she learned that she was pregnant, about to fulfill her dream of being a mother.
Imbalance the other way
I, myself, operated on the other end of the spectrum. There was a time and place in the beginning of my business that I wasn't being seen, heard, and my message was ignored.
I was disappointed that my needs felt so unmet. In fact on a financial level, I was sinking fast as I wasn't able to cover my overhead. I remember thinking that I was not making a contribution in the world, and I was beginning to doubt my abilities. I wondered if I was supposed to return to working somewhere else that guaranteed a paycheck. The whole time, I had this voice inside of me that encouraged me to awaken and remember my dreams, what created my passion to serve others, and share my story to begin with. I somehow knew my dreams were valid, and if I was going to succeed I needed help. I decided it was necessary for me to hire a mentor that was successful within a soul-aligned business themselves.
After that, I discovered I was operating so far over into the divine feminine side of flow: compassion, understanding, feelings, acceptance and holding space for others. At the same time, I didn't have structure, drive, a solid plan of action, dedication and boundaries. Without these traits, I sabotaged myself and in turn, didn't attract clients who were willing to invest in themselves. Even if they were, me being over-sensitive blinded me to the fact that they actually were ready to invest or they would not have been sent to me. I realized that by being overly nurturing and sensitive to the needs of others above my own that I was creating an abyss of hopelessness for me to sink into. I questioned my ability to show up in the world.
Aligning with what you want
Once I brought awareness into the dynamics I was operating within, I was able to shift my state of being into being more curious, motivated, and peaceful. I experienced more joy and created a more magical experience for myself. The conditions of my business, my finances, and the way I showed up for my clients improved. Over a short period of time, platforms presented themselves to me that opened up venues to share my story, reach people on a global level, and guide others into discovering more about themselves. I witnessed their growth and achievements as they aligned with their dreams.
As you can tell from the stories I shared above, exploring the inner and outer structures of the divine feminine and masculine is a vast sea of greater understanding of your inner world and outer world. It's a journey I highly recommend. The good news is awareness is the antidote to all of life's struggles, and life as a whole can always become greater than it currently is.
Kristy Lee is an author, empowerment coach, radio host, teacher, and Reiki Master Teacher who accesses the Akashic Records as a means to assist clients around the globe into finding their power and obtaining freedom.
I looked in temples, churches, and mosques.
But I found the Divine within my heart. -Rumi
Breakfast runs and holy rollers
Egg and cheese biscuits are my downfall. I don't keep biscuits in the house on purpose, but I will go out once in a while and go have one, which is what I did the other day. When I got my food, I checked my order because they already messed up by giving me a tray when I said it was to go, and then, they forgot to give me a cup so I could make my coffee. Couple that with the fact that I was already grumpy, this breakfast run was already off to an inauspicious start.
I was minding my own business making my coffee using all my concentration and focus to do so because I wasn't fully awake anyway when this random man gives me his card and invites me to his church. Another holy roller who wants to save my non-Christian soul. I thought. I almost blurted out: I'm Muslim, and I barely go to mosque, so why would I go to your church? Then, I decided to be cordial because he probably genuinely believed he should spread "the Word" to my heathen ass. I told him I was more spiritual than a follower, and I probably wouldn't come. Then, he proceeded to tell me about how his faith is spiritual and went into his story about how he was saved in 1992. He went on to say his relationship with Christ was the best thing ever, or something like that. I looked at him and told him I have a relationship with Christ, too. To which he responded, "Praise the Lord," which I returned. I told him, "Thank you, Sir" and went on my merry way.
Losing my religion
This kind of situation is why organized religion has been such a turnoff to me in my life. It always seemed like me just existing in my own space is a call to save my soul. The implication that there is only one way to God or one way to do things never sat well with me and always made my practice of religion a chore. Why would I want any parts of any institution or being that tells me I'm not good enough no matter what I do? I don't care whose religion it is. I've had a tempestuous relationship with my own religion, Islam for this very reason.
Being sin shamed at various stages added an unnecessary tension to my learning religion. I tried to do the right things. I tried to learn prayers,but it was hammered into me that if I didn't pronounce the Arabic correctly, my prayer wouldn't be accepted. Arabic is hard when English is your first language. As a language acquisition expert, I know you have to interact with a language meaningfully regularly to really master it. Outside of memorizing prayers, I had no interaction with Arabic. How am I supposed to have perfect pronunciation in that case? This idea of perfection had me nervous every time I prayed. The unsettling feeling of being wrong always came over me. It didn't always make me feel good to pray, so I didn't always do it.
Never mind the fact that I just didn't conduct myself as a "good Muslim" should. My most egregious sin from the time I was a child to even into adulthood was eating with my left hand. I had one uncle tell me the devil eats with his left hand. Another uncle told me "Muslims don't eat with their left hand." Then, an aunt noticed I was a lefty, and immediately informed me that was, haram. Sin. Let me break the judgment down. I was compared to the devil, told I wasn't Muslim, and finally told using my naturally dominant hand was a sin. All this, as I am minding my own business trying to eat.
In fact, a simple act of wearing nail polish eventually became improper and could lead me to hell. So I was told. You can't wear nail polish because it nullifies your purification ritual before you pray, and the water can't get under your nails. If you don't have your purification ritual, your prayer is not accepted. You don't pray. You go to hell. Sounds convoluted, right? I always thought so.
You can't wear shorts. If you're not covered up, you're going to hell. You shouldn't shave your legs because you're not supposed to put a blade to your body. You put a blade to your body. You're going to hell.
So if I'm going to hell no matter what I do, what's the point? Why would I want any parts of a God who is going to judge me harshly? I realized early on: it's not God judging me, it's fucking people. I don't blame my relatives or any of these other holy rollers for their programming. The bullshit that says that there is a set of rules you have to follow to get to God, and if you don't follow those rules, there is no hope for you. In their minds, they were being helpful. They were taught to fear, so that's how they tried to teach me. Fear has been a powerful tool in the teaching of religion, and it always will be.
Finding my religion
When I took the shame and fear out of religion and ritual, it became rewarding. Since I'm no longer concerned about making mistakes when I do pray, I now find prayer to be more uplifting instead of cause for anxiety. It's a more meditative process now. For me, a ritual like Hajj, the pilgrimage to Mecca that's going on this week can be enlightening if one allows it. I enjoy talking to people who have done it and what it means to them. I do believe pilgrimage to Mecca will be a part of my journey one day. Not because I have to, because I want to. When religion is not being shoved down my throat, I'm actually inclined to practice it, in moderation.
Despite well-intentioned Muslims and Christians, I don't need to be saved. I have already saved myself from rigid thinking and enslavement to ritual. I decided as long as I try to do good in the world and cause no harm, me and God are cool. For me, there is no path to God because there is no separation from God, God is in you. Unconditional love is the only path to follow. Whether you find that path through organized religion, spirituality, or your own concoction. I don't care. Just find it. I, personally find it through my own blend of religion and spirituality. Walking in unconditional love is what saved me. Not going to a church or mosque or eating with the hand I'm supposed to eat with.
Until next time, look behind, and beyond the veil...
Photo by Christopher Rusev on Unsplash
Struggle is Real
Surrendering in the middle of struggle is a beautiful thing! We are so strongly oriented to do and to push through any obstacles as we pursue big goals. Anyone that has achieved anything of significance in life has learned how to get into that type A mindset, and to just go, go, go! But we all have times when every action appears to just lead to a roadblock or dead end. At these times, actions that have previously led to accomplishment instead take us nowhere. We struggle and feel that we have struggled only to fail. When we experience this, it is important to take a step back. Realize that there is great beauty in the faith that things that are meant for us will work out eventually. I'm convinced that these moments occur for that very reason. That there is a fantastical magic to the universe, and that, often, it brings us more than we can even imagine. If this is a new way of thinking for you, I recommend exploring it.
The next time that you are taking actions, but just feeling like there is no forward movement no matter what you do: take a step back and summon your faith. There is at least a glimmer of faith in you that things will work out, and that forces of good are there to support you in your goals. As you take this step back, draw on the deep breaths that help when you are stressed. When we feel pressure, our breathing tends to be shallower than it should be. Inhale beauty and love and understand all the knowing that you need will be revealed to you in time. That surrender into faith possesses a great beauty and light to it. It might also feel tremulous and hard to hold onto when you are wanting nothing more than to achieve forward movement in your life. But see, that's why it is happening! The universe is prodding you to surrender to what is going on, just for a bit. Let things happen, and take a break from trying to control your surroundings.
Step Back
One more thing: I used to find great difficulty in getting back on task, after periods of surrender and letting go of the need to control things in my environment. These moments would sometimes stretch into hours or days, and turn into a prolonged journey into escapism. It is always freeing to step away from a hectic life. But implementing a step to stay attached to my life was key for me. You might find that this is also true for you.
My version of surrender now includes some masculine, task-oriented steps. I find these steps help keep me from getting too far into the clouds as I take these self-focused steps.
Here are the steps I recommend for cultivating a soul-directed state of surrender:
1. List out your big goals in life
Start working to align your daily schedule to these goals. Commit to 1 day a week (or every 2 weeks, based on what your current schedule and responsibilities allow) where you let go of any to do's that are not urgent and not related to these big goals. Revisit this process a few times each year.
2. Declutter an area in your house, car, or at your place of work.
This usually brings a quick burst of energy and allows for remembrances of the past and enhancing creativity. Keep in mind that you are doing this activity just for yourself, and not to impress anyone.
3. Take at least a day to explore what you are drawn to, in terms of food, activities, locations and people.
You will see great things as you follow these steps. Often, a new path or a new way of doing things will start to show up. It might appear as the seed of an idea that grows stronger in definition. The above steps will encourage this seed's growth into larger ideas. From these ideas, you can formulate concrete actions, and break them down into smaller steps.
I recommend trying different variations to see what works best based on your own natural tendencies, habits and regular daily schedule. Maybe you are overly task-oriented, and could really use the extra time to reflect on what your soul desires. Or, maybe you are like me and prefer a somewhat regimented form of surrender to keep you grounded. As you try different things, you will find a combination that works best for you. With practice, you can turn surrender into a powerful tool that will allow for beautiful awarenesses and deeper moments of pleasure in your life.
Follow Your Own Path
You will see great things as you follow these steps. Often, a new path or a new way of doing things will start to show up. It might appear as the seed of an idea that grows stronger in definition. The above steps will encourage this seed's growth into larger ideas. From these ideas, you can formulate concrete actions, and break them down into smaller steps.
I recommend trying different variations to see what works best based on your own natural tendencies, habits and regular daily schedule. Maybe you are overly task-oriented, and could really use the extra time to reflect on what your soul desires. Or, maybe you are like me and prefer a somewhat regimented form of surrender to keep you grounded. As you try different things, you will find a combination that works best for you. With practice, you can turn surrender into a powerful tool that will allow for beautiful awarenesses and deeper moments of pleasure in your life.
Amy Mehta Parmar is a Reiki master and transformational women's empowerment coach. Her digital course, 'Ditch the baggage and raise your vibration' is a great option for anyone that wants to get rid of emotional luggage that has followed them around and, at times, poisoned their relationships with others. She can be found at www.amymehta.com
Photo by Ross Sokolovski on Unsplash
All Dogs Go to Heaven
Recently, something happened in my old neighborhood that made me think about how protective I can be over who I love. Two large dogs got away from their owner during a walk. They attacked and killed a smaller senior dog who just happened to be in the wrong place at the wrong time on his walk. I wasn't there, but I imagined everything happened so fast that neither owner had time to prevent anything from happening. It was just very sad. It made me think of a time when I unequivocally decided I would willingly be mauled to protect my dog, Edison.
When I used to walk him in my old neighborhood, I used to take him to a beautiful neighborhood across the street. Houses of different styles, shapes, and sizes, not cookie cutter style homes you see in a lot of developments nowadays. Two of the houses had ponds on the edges of their properties. Near one of the ponds, a family of Canadian geese claimed their squatter's rights. Edison tried to bird dog the geese many times, but considering it was private property, I didn't let him. This curbing of his natural instincts aside, we both loved this walk. It was just a beautiful old school neighborhood.
The Wanderer
Apparently, we weren't the only ones who enjoyed the natural views. One day, I took Edison for his usual morning walk. We meandered on our usual route when we saw a four-legged creature some 200 feet away. Sporting no collar that I could see, it had pointed ears, a bushy tail, and a sly, slithering gait. Clearly, not the run of the mill, neighborhood dog.
This was not the first time I had seen this creature. Two weeks before that, I saw him sneaking around near a different house, trying to blend in. My first guess was that it was a fox, but the second time I saw him I realized he was too big and too beige to be a fox. I looked at him. He looked at me, and we both went about our business. After further introspection, I realized this intrepid wanderer was a coyote.
My second encounter with Cruiser (the name I randomly assigned him) was a little more precarious because this time, I had Edison with me. The three of us looked at each other and stopped in our tracks. Edison is a sociable spirit. His inclination was to embark on a doggy meet and greet. My instinct was for the preservation of my dog and myself.
Without sudden movement that could be mistaken for aggression, I steered Edison away from his wild cousin. (Don't all Indian parents do that?) He didn't get it at first. With his usual, excited smile, his look said, "Why aren't we going that way?" I talk to my dog like he's human, so I said, "We are not going over there because he will fuck you up!" He still didn't seem to get it, so I repeated, "You don't understand! He will fuck you up!" I believe my dog read my nervous energy and had heard me say fuck enough times to know that he should go with Mommy on this one.
We continued in the opposite direction. Cruiser still stared at us. Edison didn't have a care in the world but I thought to myself: Please God, don't let this animal go after my dog. Visions of being in an emergency room because the coyote mauled me floated in my head. Not how I would want to spend a day. But I made the decision to stand my ground and keep this animal away from Edison. Emergency room or not. Coyote or Chihuahua, I'm not letting anything attack my dog. I turned around again and Cruiser went on his merry way.
I never saw him again after that. To this day, I don't know what happened to Cruiser or what his business was in that neighborhood. I think he was casing it for something. I don't know if he had a wife, kids or if he was on his own just looking for food. As long as he didn't try to get Alpha on Edison or make him his next meal, I decided to be cool with him and not go ugly on the coyote.
Protecting Who We Love
As I recalled this amusing incident with Edison and how protective I am of him, I realized I am a protector of those I love. Whether it's my mother, my nieces and nephews, friends, or my dog, I have a natural instinct to keep them safe from harm, emotional or physical. If I am inclined to love you, I am inclined to protect you. It goes hand in hand.
Not that I look for fights or any other sort of chaos, but I will be a warrior for someone I love. I will stand for you and beside you. At the same time, I will actively avoid destructive action, but if I have to, I will mess some shit up. That would be in a rare circumstance, though. At the end of the day, just like Cruiser, I want to be a happy traveler, going my own may.
Until next time… look behind and beyond the veil…
Photo by Kalen Emsley on Unsplash
The Blonde and the Brown
A few nights ago, I was out watching a band. My view was somewhat obstructed because I was sitting at the bar, and, instead of being onstage, the singer spent a lot of time working the crowd. But his voice was unmistakable. It had that rock n' roll growl that I like. I was sitting there trying to enjoy the show when an inebriated Heather Locklear wannabe with too much lip filler kept accosting me and my friend with her deep knowledge of Indian culture.
She became offended when we had the audacity to disagree with her take and pretty much had no fucking idea what she was talking about. We both tried to diffuse her and ignore her for the most part. She sat right next to me, so she kept trying to engage me in conversation which was part her to trying justify herself and part her trying to engage me in inane chit-chat.
She asked me how old I was, and I told her I am 44. "You don't look it." Immediately followed that bit of demographic information. Then, "Do you have kids?" After I responded with a matter-of-fact, "No." Her next question was, "Are you a lesbian?" To which, I responded with, "No, I just didn't want to have kids with just anybody." Then, she agreed with me and affirmed that it's best not to have kids if you don't want them, as if I needed her to sign-off on that. Her ridiculous question and ensuing comments instantly squashed any inclination to keep placating her. Then I thought. Wow. You're ignorant. I ignored her after that.
The Runaway Bride
First of all, why would not having kids make someone a lesbian? As if your sexual orientation has anything to do with it. Some lesbians do choose to have children. That's exactly what having children is: a choice. Unless I'm asking you to help me raise them, whether or not I have children is a choice that has nothing to do with you.
I don't have children right now because I want to give them the best I can possibly give them at the start. At the least, I can choose a partner who loves me and helps me grow, and I would do the same for him. That is the relationship model I want to give them. If I had them before now, I would not have been able to give them that.
I have been in love twice in my life. Both times, I was the runaway bride. When I knew a relationship wasn't going to keep me happy for the rest of my life, I bailed. I had no intention of potentially fucking up at least two peoples' lives if I went against my own intuition. I didn't want to get divorced, or worse, stay in something that doesn't work because I was afraid to be alone. I never was afraid to be alone, and I never will be. I would rather be happy, free, and on my own instead of miserable, with someone just because that's what's expected, and trapped. Shame on me, if I would ever bring children into something I knew would fail to start with.
Happy and Free, So Let Me Be
I know a happy medium exists, and I know I will find it. I'm not going to let someone else define what that is and what should fulfill me as a woman. I'm lucky to live in a time where perspectives on the roles of women are expanding. It's not just daughter, sister, wife, or mother, who you are to someone else that defines your womanhood. It's more who you are for yourself and defined by yourself. You can mix all the other stuff in as you choose.
Right now, I do things that make me happy all day. My work is my passion. I decide my own schedule. I live the most authentic and organic life I can possibly live. I am the best I have ever been. Emotionally, I am in the best position to have children. However that happens is for me and the Divine to decide. Not some plastic shit-stirrer at a bar.
Until next time... look behind and beyond the veil...
Sameena K. Mughal, Author, Freelance Writer
October 2020


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Beginning June 28, the art exhibit "Constellation" will be the coolest thing on the Hudson River. The best way to view the striking installation—comprised of 17 tapered aluminum poles holding LED globes up to 80 feet above the Bannerman Castle ruins—will be from the water. So book a wild three-hour excursion at sunset to Bannerman Island with Storm King Adventure Tours.
Visit melissamcgillconstellation.com for more info.


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10K Bonus based on eligibility
Clinical Coordinator Full Time
Coordinates functions and activities related to a single departmental function. Position is responsible for a clinic unit. Position coordinates the scheduling efforts of the unit RN's. Acts as liaison person to internal and external customers (i.e., pre-hospital providers, medical and nursing staff, hospital administration). Responsible for developing and implementing plans/processes for meeting regulatory requirements. Trains and mentors staff.
Special Skills: Effective interpersonal and communication skills. Ability to effectively implement change and assist with the management of departmental budget. Must be able to independently make decisions in high-stress situations.
Minimum Qualifications: Nursing Degree from an accredited nursing school. Two years relevant clinical nursing experience.
Preferred Qualifications: BSN. Management experience with two years supervisory. Certification in specialty area of practice preferred..
Required Certifications/Licensure:
ER, ICU, Endoscopy, Tele, Cardiac Cath Lab, L&D: ACLS required upon orientation completion.
Job: Nursing Assistants and PCA
Primary Location: Phoenix, Arizona
Facility: Abrazo Central Campus
Job Type: Full-time
Shift Type: Nights
Employment practices will not be influenced or affected by an applicant�s or employee�s race, color, religion, sex (including pregnancy), national origin, age, disability, genetic information, sexual orientation, gender identity or expression, veteran status or any other legally protected status. Tenet will make reasonable accommodations for qualified individuals with disabilities unless doing so would result in an undue hardship.
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Society of Gastroenterology Nurses and Associates
330 N Wabash Ave. Suite 2000, Chicago IL 60611
800.245.7462, in Illinois - 312.321.5165
Fax: 312.673.6694 | firstname.lastname@example.com
Society of Gastroenterology Nurses and Associates
330 N Wabash Ave. Suite 2000, Chicago IL 60611
800.245.7462, in Illinois - 312.321.5165
Fax: 312.673.6694 | firstname.lastname@example.com