To Life
"Saving a Life (Pikkuah Nefesh)
Most of Jewish law can and should be set aside in order to avoid
endangering a
person's health or safety.
By Rabbi Simon Glustrom
Every effort must be made to save life, according to Jewish law,
unless it
involves violating the cardinal negative precepts of murder,
idolatry, incest,
or adultery. The talmudic rabbis interpret the verse "You shall not
stand idly
by the blood of your neighbor" (Leviticus 19:16) to mean that if a
person is in
danger of drowning, it is the duty of all who can swim to dive in to
save him.
This article explores more commonplace conflicts between the value
of preserving
life and other demands of traditional Jewish norms. Reprinted with
permission
from The Language of Jewish Life (Jason Aronson).
The preservation of human life takes precedence over all the other
commandments
in Judaism. The Talmud emphasizes this principle by citing the verse
from
Leviticus [18:5]: "You shall therefore keep my statutes…which if a
man do, he
shall live by them." The rabbis add: "That he shall live by them,
and not that
he shall die by them." (Babylonian Talmud, Yoma 85b)
When life is involved, all Sabbath laws may be suspended to
safeguard the health
of the individual, the principle being pikkuah nefesh doheh Shabbat--
[rescuing
a] life in danger takes precedence over the Sabbath.
One is not merely permitted--one is required to disregard a law that
conflicts
with life or health. "It is a religious precept to desecrate the
Sabbath for any
person afflicted with an illness that may prove dangerous; he who is
zealous is
praiseworthy while he who asks questions sheds blood." (Shulhan
Arukh, Orah
Hayyim 328:2)
This duty to ignore the law, if necessary, to safeguard health is
also stressed
in connection with fasting on Yom Kippur. A sick person is obliged
to break the
fast. Neither the patient nor those attending him need atone when
performing
such acts that are forbidden under normal circumstances.
In spite of the virtue of observing a fast, it is not virtuous to
observe laws
at the risk of one's life. Such conduct is regarded as foolish, even
as sinful.
The Sages described this stubbornness as a "piety of madness."
Pikkuah nefesh was not only confined to serious crises in health.
The victims of
religious persecution who lived under constant threat of death were
also guided
by the principle of pikkuah nefesh. They, too, were cautioned against
sacrificing their lives in order to observe the Sabbath and
festivals. There
were exceptions, to be sure, when martyrdom was considered the
greater virtue
than surrendering one's principles. Generally, however, the Jew was
encouraged
to accept temporary indignity and choose life, to live in misery,
rather than to
die in glory.
Simon Glustrom is rabbi emeritus of the Fairlawn Jewish Center in
Fairlawn, New
Jersey, and a past chairperson of the Rabbinical Assembly's
Publications
Committee. He is the author of When Your Child Asks: A Handbook for
Jewish
Parents (Bloch Publishing Co.) and The Myth and Reality of Judaism:
82
Misconceptions Set Straight (Behrman House)."
The article below is dated 07/14/2005 (c.e.)
"Green tea and cancer: A mixed bag
By Bernadine Healy, M.D.
Green tea has been a medicinal potion for thousands of years. Laden
with plant chemicals called flavonoids known for their powerful
antioxidant abilities, green tea is touted to protect against two of
the biggest of human scourges—coronary disease and cancer. But just
how green tea works its wonders in the prevention or treatment of
individual disease remains a mystery. Thomas Gasiewicz, researcher
in the Department of Environmental Medicine at the University of
Rochester Medical Center, presented evidence for some of its magic
at an international conference on diet and cancer held in
Washington, D.C., today.
His laboratory demonstrated that the prime antioxidant component of
green tea, which is in the family of plant chemicals called
catechins—or epigallocatechin-3-gallate (EGCG) to be precise—zooms
in on a key target in the cancer cell. And the target is a big one:
a normal stress protein, known as heat shock protein 90 (HSP90).
Heat shock or stress proteins are critical to survival of all cells,
cancerous or otherwise.
Stress proteins abound in both plants and animals. Think of them as
protectors that chaperone the thousands of worker-bee proteins that
interact in and on the surface of our cells in the course of any one
cell's life. Growth, performance, communication, you name it, and
some form of HSP is a key player. When cells are threatened by a
treacherous environment such as heat (from which we get the name
HSP), proteins curl up and then clump up. We now know it also
happens with damaging cold, low oxygen, or poisons. Heat shock
protein protectors quickly rev up and come to the rescue to both
repair injured proteins and to carry the irreversibly damaged ones
to a disposal dump for an out-of-the-way burial so new ones can take
their place.
Cancer hijacks the stress protein network in its efforts to overtake
the body. Cancer cells are fast growing and on the march wherever
they set up shop—breast, prostate, colon, bone marrow. And in that
superstressed state of attack, cancer cells produce abnormally high
levels of HSP90 to protect their cancer-producing proteins. Even in
the face of toxic radiation and chemotherapy, some cancer cells
survive because of these natural potent protectors. What Gasiewicz
and his colleagues have shown is that the age-old EGCG does battle
with HSP90. A few months ago, his laboratory reported for the first
time that EGCG binds to this protective stress protein important to
cancer growth and survival and essentially takes it out of
commission.
One caveat: You have to drink a lot of tea to get enough EGCG to do
any good. Just how much is not known, but it is somewhere between
three to 10 cups a day. Because of its bitterness and the caffeine
load of that much tea, that's hard to do. No wonder that after a
detailed analysis of numerous studies of green tea and cancer
involving tens of thousands of patients, the Food and Drug
Administration announced only two weeks ago that there is "no
credible evidence" to support green tea's health claims when it
comes to cancer. And for the two cancers where the studies are the
most promising, namely prostate cancer and breast cancer, the FDA
calls it "highly unlikely" that green tea reduces the risk of
either. However—and there's always a however in clinical medicine—
studies are underway to look at cancer and concentrated green tea
extract, which is rich in ECGC but free of the side effects of
overdosing on the full tea brew. Email your comments or suggestions
to
healthbriefs@... "
"Scientists shed new light on ageing process
June 30 2005 (c.e.)By Tan Ee Lyn
Hong Kong - Scientists in Hong Kong have shed new light on why cell
repair is
less efficient in older people after a breakthrough discovery on
premature
ageing, a rare genetic disease that affects one in four million
babies.
Premature ageing, or Hutchison-Gilford Progeria Syndrome (progeria),
is obvious
in the appearance of a child before it is a year old. Although their
mental
faculties are normal, they stop growing, lose body fat and suffer
from wrinkled
skin and hair loss.
Like old people, they suffer stiff joints and a build-up of plaque
in arteries
which can lead to heart disease and stroke. Most die of
cardiovascular diseases
before they are 20.
In 2003, a team of scientists in the United States found that
progeria was
caused by mutation in a protein called Lamin A, which lines the
nucleus in human
cells.
A team at the University of Hong Kong, led by Zhou Zhongjun, took
the research a
step further in 2004 and found that mutated Lamin A actually
disrupted the
repair process in cells, thus resulting in accelerated ageing.
The study was published in the July issue of the Nature Medicine
journal.
Zhou said the team came by their findings after comparing skin cells
taken from
two progeria sufferers, normal humans, progeria mice and normal mice.
While damaged DNA was quickly repaired in the healthy human and mice
cell
samples, the samples taken from the progeria humans and mice had
difficulty
repairing damaged DNA.
"Mutation in this protein (Lamin A) can cause defects in repair and
thus lead to
progeria," Zhou, a research assistant professor with the
biochemistry department
at the University of Hong Kong, said in an interview.
"DNA damage is not effectively repaired in cells with defective
Lamin A but very
efficiently repaired in normal cells."
The study highlights the importance of Lamin A to the repair
process, and any
mutation to Lamin A that disrupts repair will bring about ageing,
Zhou said.
Having established the link between Lamin A and repair, Zhou is
using major
findings from other research he did in 2002 to work on his next
project, a
product which he hopes could kill cancer cells.
Zhou, Professor Karl Tryggvason in Sweden's Karolinska Institute and
a Spanish
research group found in 2002 that the enzyme Zmpste 24 was
responsible in
converting prelamin A to functional Lamin A.
Zhou's laboratory is now developing inhibitors to Zmpste 24, which
he hopes to
apply to tumours. These inhibitors should theoretically disrupt
Lamin A
production, thwart the repair function in cancer cells, and bring on
their
premature aging and death.
"We're now trying to develop inhibitors to Zmpste 24 and apply it to
tumour
cells," he said."
"L'CHAIM-TO LIFE!"
With this post we continue a series of articles on health from
various sources
which we hope will stimulate more research and
conversation in keeping with the Jewish tradition "L'CHAIM-TO LIFE!"
"Dietary Curcumin Inhibits Chemotherapy-induced Apoptosis in Models
of
Human Breast Cancer
http://cancerres.aacrjournals.org/cgi/content/full/62/13/3868
[Cancer Research 62, 3868-3875, July 1, 2002]
2002 American Association for Cancer Research
Tumor Biology
Dietary Curcumin Inhibits Chemotherapy-induced Apoptosis in Models of
Human Breast Cancer
1
Sivagurunathan Somasundaram, Natalie A. Edmund, Dominic T. Moore,
George W. Small, Yue Y. Shi and Robert Z. Orlowski2
The Lineberger Comprehensive Cancer Center [S. S., N. A. E., D. T.
M.,
G. W. S., Y. Y. S., R. Z. O.] and the Department of Medicine [R. Z.
O.], Division of Hematology/Oncology, The University of North
Carolina
at Chapel Hill, Chapel Hill, North Carolina 27599
Curcumin, the major component of the spice turmeric, is used as a
coloring and flavoring additive in many foods and has attracted
interest because of its anti-inflammatory and chemopreventive
activities. However, this agent also inhibits the generation of
reactive oxygen species (ROS) and the c-Jun NH2-terminal kinase (JNK)
pathway, and because many chemotherapeutic drugs generate ROS and
activate JNK in the course of inducing apoptosis, we considered the
possibility that curcumin might antagonize their antitumor efficacy.
Studies in tissue culture revealed that curcumin inhibited
camptothecin-, mechlorethamine-, and doxorubicin-induced apoptosis of
MCF-7, MDA-MB-231, and BT-474 human breast cancer cells by up to 70%.
Inhibition of programmed cell death was time and concentration
dependent, but occurred after relatively brief 3-h exposures, or at
curcumin concentrations of 1 µM that have been documented in Phase I
chemoprevention trials. Under these conditions, curcumin exhibited
antioxidant properties and inhibited both JNK activation and
mitochondrial release of cytochrome c in a concentration-dependent
manner. Using an in vivo model of human breast cancer, dietary
supplementation with curcumin was found to significantly inhibit
cyclophosphamide-induced tumor regression. Such dietary
supplementation was accompanied by a decrease in the activation of
apoptosis by cyclophosphamide, as well as decreased JNK activation.
These findings support the hypothesis that dietary curcumin can
inhibit chemotherapy-induced apoptosis through inhibition of ROS
generation and blockade of JNK function, and suggest that additional
studies are needed to determine whether breast cancer patients
undergoing chemotherapy should avoid curcumin supplementation, and
possibly even limit their exposure to curcumin-containing foods.
Curcuma longa Linn is a perennial herb originally cultivated widely
in
tropical regions of Asia from which dried rhizome is isolated the
spice turmeric (reviewed in Refs. 1, 2, 3 ). Curcumin, also known as
diferuloyl methane, is the major yellow pigment extracted from
turmeric, which is used extensively in curries. Its properties as a
coloring and flavoring agent have led to uses as a dietary additive
in
a variety of foods (1 , 3 , 4) . These include saffron, mustard and
other spices, gelatins, puddings, sorbets, ice creams, soups, meats,
pickles, margarine, and both alcoholic and nonalcoholic beverages.3
Extracts containing curcumin have also been used in medicines in
India
and Southeast Asia for generations, and according to tradition are
useful in the treatment of inflammation, skin wounds, hepatic and
biliary disorders, cough, and coryza, as well as certain tumors (1 ,
3
, 4) . As a result, dietary intake of curcumin is especially high in
these areas of Asia, where adults consume up to 200 mg of
curcumin/day
or up to 7.8 µmol/kg of body weight (4) . Even in France, however,
where curcumin exposure may be more representative of that typical
worldwide, intake of as much as 3.4 µmol/kg/day has been documented
(5) .
The exposure of populations worldwide to curcumin, and its many uses,
has led to studies aimed at elucidating some of its activities.
Curcumin and related compounds inhibit free radical generation and
act
as free radical scavengers (6, 7, 8) and antioxidants (9) ,
inhibiting
lipid peroxidation (10 , 11) and oxidative DNA damage (12) .
Inhibition of lipoxygenase and cyclooxygenase (13) resulting in
decreased arachidonic acid release and metabolism, along with
abilities to inhibit activation of NF-B4 (14 , 15) , may contribute
to
the anti-inflammatory activity of these compounds. Another property
ascribed to curcumin is that of inhibition of c-jun/AP-1 function
(16)
and JNK activation (17) . Curcuminoids have been noted to be potent
inhibitors of cytochrome P450 (18) and to have the ability to induce
glutathione S-transferase (19) , and as such, have been proposed as
potential chemoprotective agents (reviewed in Ref. 20 ). Because
curcumin inhibits tumor formation in several murine tissues and
antagonizes both initiation and promotion of tumors in rodent
epithelial and colon cancer models (1, 2, 3) , interest has been
raised in this compound as a chemopreventive agent (reviewed in Ref.
21 ). Most recently, curcumin has demonstrated antiangiogenic
properties in several laboratory and in vivo model systems (22, 23,
24) . These properties of curcumin have led to several Phase I human
trials that have shown this agent to be tolerated well (25 , 26) ,
and
their successful completion suggests that curcumin use may increase
in
the future.
Curcumin's chemopreventive activity in animal model systems has led
investigators to study its potential impact upon tumor cell growth
and
apoptosis. Several reports document an antiproliferative effect on
cultured cells such as on colon cancer (27) and breast cancer cells
(28) . This may, in part, be because of programmed cell death because
at high concentrations curcumin can induce apoptosis such as in human
leukemia cells (29) . In contrast, in other systems curcumin can
inhibit apoptosis such as in T lymphocytes (30) , and it protected
rat
lungs from injury by bleomycin (31) and rat myocardium from
Adriamycin
(32) , respectively, but its impact on the therapeutic applications
of
antineoplastic drugs has not been well studied. Because ROS have been
felt to play important roles in drug-induced apoptosis (reviewed in
Ref. 33 ), one might suspect that curcumin, as an antioxidant and
free
radical scavenger, would inhibit the ability of chemotherapeutic
drugs
to induce apoptosis. Furthermore, curcumin inhibits JNK activation
(17) , which has been associated with chemotherapy-mediated induction
of apoptosis in tumor cells (reviewed in Ref. 34 ). We therefore
considered the possibility that curcumin might decrease the
effectiveness of antitumor drugs, and we used breast cancer as an
example of the possible systemic effects of this dietary compound.
Here we report, using both tissue culture and in vivo models of human
breast cancer, that curcumin inhibited the proapoptotic activity of
several chemotherapeutic agents and also inhibited ROS generation,
JNK
activation, and release of mitochondrial cytochrome c. These findings
support the need for further study into the effects of dietary
curcumin on patients receiving breast cancer chemotherapy, and may
indicate a need for such patients to avoid dietary supplementation
with curcumin and perhaps even limit their intake of foods containing
this agent.
Chemicals.
Curcumin, mechlorethamine, and cyclophosphamide were from Sigma
Chemical Co. (St. Louis, MO), whereas camptothecin and doxorubicin
were obtained from Calbiochem-Novabiochem Corp. (San Diego, CA).
Stock
solutions were prepared in 95% ethanol (mechlorethamine; Mallinckrodt
Baker, Inc., Paris, KY), PBS (cyclophosphamide; LCCC TCF), or DMSO
(curcumin, camptothecin, and doxorubicin) and stored at -20°C. These
agents were added to the concentrations indicated, with a final
vehicle concentration of 0.5% (v/v). Aprotinin and leupeptin were
from
Roche Molecular Biochemicals (Indianapolis, IN). All other chemicals
used were from Fisher Scientific (Fair Lawn, NJ).
Cell Lines and Cell Culture.
MCF-7, MDA-MB-231, and BT-474 human breast carcinoma cells (LCCC TCF)
were propagated in Richter's modified Eagle's medium supplemented
with
insulin, gentamicin, and 20 mM HEPES (LCCC TCF), and 9% FCS,
penicillin G sodium at 100 units/ml, and streptomycin sulfate at 100
µg/ml (Life Technologies, Inc., Grand Island, NY).
Apoptosis Assays.
To directly visualize apoptosis-associated DNA fragmentation, 3.0–4.0
x 106 cells subjected to conditions described in the text were
detached, and their supernatants were collected by centrifugation.
Pellets were resuspended in 10 mM Tris-HCl (pH 8.0), containing 10 mM
EDTA and 0.5% Triton X-100, and lysed by vortexing, and debris was
removed by centrifugation. After extraction with
phenol-chloroform/isoamyl alcohol, nucleic acids were ethanol
precipitated, collected by centrifugation, redissolved in 10 mM
Tris-HCl (pH 8.0), containing 1 mM EDTA, 50 mM NaCl, and DNase-free
RNase (Roche Molecular Biochemicals), and incubated at 37°C for 2 h.
DNA separated in 1.5% agarose gels was visualized by staining with
ethidium bromide.
For quantitative assessment of oligonucleosomal DNA fragmentation,
medium containing the agents tested was added to 1.2 x 105 cells, and
apoptosis was detected using the Cell Death Detection ELISAPLUS kit
(Roche Molecular Biochemicals). Spectrophotometric data at a
wavelength of 405 nm, with a reference of 490 nm, were acquired on a
MAXline Vmax kinetic microplate reader (Molecular Devices
Corporation,
Sunnyvale, CA). The enhancement of apoptosis was calculated in
relation to control cells receiving vehicle alone and tabulated in
KaleidaGraph version 3.0.1 (Synergy Software, Reading, PA).
To perform caspase 3 activity assays, 1.0 x 106 cells exposed to
conditions detailed subsequently were harvested, collected by
centrifugation, and lysed with 10 mM Tris-HCl (pH 7.5), containing 10
mM sodium phosphate, 135 mM NaCl, 1% Triton X-100, 10 mM Na PPI, 1 mM
phenylmethylsulfonyl fluoride, 1 µg/ml aprotinin, and 1 µg/ml
leupeptin, for 10 min at 4°C. The lysates were clarified by
centrifugation, aliquots were set aside for protein concentration
determinations using the BCA assay (Pierce Chemical Co., Rockford,
IL), and caspase 3 activity was evaluated using the substrate
acetyl-aspartyl-glutamyl-valyl-aspartate (PharMingen, San Diego, CA).
Release of the 7-amino-4-methyl-coumarin was determined by
fluorescence spectrophotometry using an excitation wavelength of 380
nm, and an emission wavelength of 460 nm, and a Perkin-Elmer LS50B
luminescent spectrometer (Perkin-Elmer Instruments, Shelton, CT). The
ratio of caspase 3 activity was calculated in relation to control
cells, which received vehicle alone.
ROS Assay.
Cells (1.0 x 106) exposed to conditions detailed below subsequently
were washed with ice-cold PBS, detached, collected, resuspended in
PBS, and transferred to 96-well plates in triplicate. Fifty µl of a
16
mM solution of dichlorodihydrofluorescein diacetate (Sigma Chemical
Co.) in PBS was then added, and the plates were incubated at 37°C for
1 h. Fluorescence at an emission wavelength of 485 nm, after
excitation at 530 nm, was assayed as described above. An aliquot of
the cell suspension was lysed and protein concentrations were
determined. The final results were expressed as arbitrary absorbance
units/mg protein.
JNK Assays.
JNK activity, as judged by the presence of phosphorylated c-Jun
protein, was determined with the Trans-AM. AP-1 c-Jun ELISA kit,
which
was used according to the manufacturer's specifications (Active
Motif,
Carlsbad, CA). Briefly, AP-1 heterodimeric complexes from nuclear
extracts were captured by binding to a consensus 5'-TGA(C/G)TCA-3'
oligonucleotide immobilized on a 96-well plate. The phospho-c-Jun
content of the bound AP-1 was determined in a colorimetric reaction
using a phospho-c-Jun primary antibody and a secondary horseradish
peroxidase-conjugated antibody. Spectrophotometric data were
expressed
as a ratio of absorbance of each experimental condition compared with
control cells exposed to vehicle alone.
Cytochrome c Release Assay.
Cells (1.0 x 106) were exposed to conditions described in the text,
collected, and homogenized on ice. The resultant lysate was clarified
by centrifugation, and aliquots containing the cytosolic fraction
were
set aside for protein concentration measurements. Samples were mixed
with 6x SDS sample buffer containing mercaptoethanol, denatured and
reduced by heating, and subjected to SDS-PAGE. After separation, the
proteins were electrophoretically transferred to nitrocellulose
filters and subjected to Western blotting with murine monoclonal
antibody 7H8.2C12 to cytochrome c (PharMingen). Immunoreactive
protein
bands were detected using the Phototope-Horseradish Peroxidase
Western
detection kit (Cell Signaling Technology, Inc.). To quantify protein
bands, autoradiographs were scanned into Adobe Photoshop 5.0 (Adobe
Systems, Inc., San Jose, CA), and densitometry was performed using
NIH
Image version 1.61 by laboratory members not involved in this project
who were blinded to the experimental conditions.
Tumor Xenograft Modeling.
All experiments were performed under a protocol approved by the
University of North Carolina at Chapel Hill's Institutional Animal
Care and Use Committee. Mycoplasma-free MCF-7 and BT-474 cells were
injected s.c. in the flanks of nu/nu mice (Charles-River
Laboratories,
Inc., Wilmington, MA). Animals receiving MCF-7 cells had been
implanted 24 h earlier interscapularly with a pellet of 17ß-estradiol
(Innovative Research of America, Sarasota, FL). Once palpable tumors
developed, their bi-directional dimensions in millimeters were
measured using calipers, and tumor weights in milligrams were
calculated using the formula for a prolate ellipsoid, (L x W2)/2,
where L is the longer of the two dimensions. When tumors of 100-mg
size developed, the mice were randomized to either a control diet
(Lab
Isopro RMH 3000; PMI Nutrition International, Inc., Brentwood, MO) or
diets supplemented with curcumin. One day later, they received i.p.
injections with filter-sterilized PBS or an equivalent volume of PBS
containing cyclophosphamide. The impact on tumor weights was
monitored
for another 48 h with data expressed as a fold-change from day 0 for
each animal. All measurements were performed by a technician who was
blinded to the treatment assignments. The fold tumor weight
differences between the curcumin diet and regular diet groups were
compared for the fold differences from day 0 to day 2 and for the
fold
differences from day 1 to day 2 with the robust nonparametric
Wilcoxon
Two-Group test using normal scores.
Immunohistochemistry.
Three mice from each treatment assignment in a separate group of
animals were euthanized using guidelines of the American Veterinary
Medical Association's Panel on Euthanasia. Tumors were then excised
and fixed in Tissue-Tek O.C.T. (Sakura Finetek USA, Inc., Torrance,
CA) while frozen in liquid nitrogen. For detection of
apoptosis-associated ssDNA generation (35) , sections were washed in
6:1 methanol:water, air dried, and treated with proteinase K (USB
Corp., Cleveland, OH). The slides were then rinsed and heated at 56°C
in prewarmed 50% formamide (USB Corp.), followed by rinsing in
ice-cold PBS. After blocking, they were stained with the murine
monoclonal antibody Mab 3299 (Chemicon International, Temecula, CA),
with the secondary antibody consisting of cy3 fluorescent-conjugated
goat antimouse antibody (Chemicon International). Sections were
incubated for 15 min, washed in PBS, and mounted with a
4',6-diamidino-2-phenylindole nuclear stain (Vector Laboratories,
Burlingame, CA). Slides were visualized using an UV Zeiss Axioplan
fluorescence microscope (Carl Zeiss Optical, Inc., Chester, VA).
Separate photographs were taken with appropriate filters for blue
nuclear staining and red ssDNA staining, overlayed using Adobe
Photoshop software, and displayed as a fusion image at x10. The
quantification of pixels was performed using NIH Image version 1.61
with uncalibrated absorbance settings and expressed as the mean ± SE
from five measurements in each section. Immunofluorescence for JNK
was
performed in a similar fashion using the murine monoclonal G9
antibody
to phospho-SAPK/JNK (Cell Signaling Technology, Inc.).
Curcumin and Camptothecin-induced Apoptosis.
To explore the possibility that curcumin could compromise the
proapoptotic activity of chemotherapy, studies were pursued initially
with MCF-7 human breast carcinoma cells and camptothecin. This
topoisomerase 1 inhibitor is cytotoxic to breast cancer cells in
animal tumor models using patient-derived cell lines (36) , and
generates ROS (37) and activates JNK during its induction of
apoptosis
(38) . Mock-, vehicle-, and curcumin-treated cells demonstrated few,
if any, low molecular weight apoptosis-associated DNA fragments and,
whereas camptothecin induced large amounts of oligonucleosomal
laddering, in the presence of curcumin, this appeared to be
significantly blunted (Fig. 1A) . A quantitative evaluation was then
sought using an ELISA to detect histone-associated oligonucleosome
DNA
fragments. Compared with vehicle-treated cells, camptothecin induced
a
5.40 ± 0.56-fold increase in this apoptosis-associated parameter, but
in the presence of curcumin, this increase was only 1.46 ± 0.12-fold
(Fig. 1B) . Because curcumin has been reported to block DNA
fragmentation without interfering with apoptosis in T lymphocytes
(30)
, caspase 3 activity assays were performed as an independent
assessment of apoptotic changes. Camptothecin induced a 2.69 ±
0.12-fold increase in caspase 3 activity compared with the vehicle
control, but with curcumin present this increase was only 1.53 ±
0.14-fold (Fig. 1C) . Curcumin itself was able to decrease basal
caspase 3 activity in this assay to 0.65 ± 0.05-fold of control
levels, further demonstrating its ability to inhibit
apoptosis-associated events in this cell line.
Fig. 1. Curcumin inhibits camptothecin-induced apoptosis in MCF-7
cells. A, MCF-7 breast carcinoma cells were exposed for 12 h to
either
vehicle (Lane 3), 10 µM curcumin (Lane 4), 10 µM camptothecin (Lane
5), or both camptothecin and curcumin (Lane 6), and compared with
mock-treated cells (Lane 2). Apoptosis-associated DNA fragmentation
was evaluated by agarose gel electrophoresis with digested with
HindIII (Lane 1) and a 100-bp ladder (Lane 7) as standards. B, after
MCF-7 cells were treated with vehicle, curcumin, camptothecin, or
curcumin and camptothecin, apoptosis was evaluated using the Cell
Death Detection ELISAPLUS kit. Results are the mean and SE from four
experiments expressed as the fold increase in apoptosis compared with
the vehicle control, arbitrarily set at 1.00. C, caspase 3 activity
assays were performed on MCF-7 cells using the substrate
acetyl-aspartyl-glutamyl-valyl-aspartate. Results are the mean ± SE
from three experiments, expressed as the ratio of absorbance of each
sample to that of the vehicle control, arbitrarily set at 1.00. D,
time dependence was studied by treating MCF-7 cells with 10.0 µM
camptothecin for 12 h. Curcumin was either preincubated for 3 h and
then present throughout camptothecin treatment for a total of 15 h,
added at the same time as camptothecin, or at 3-h intervals
thereafter. The percentage of inhibition of apoptosis was calculated
as [(fold induction of apoptosis by camptothecin alone - fold
induction in the presence of curcumin) / (fold induction of apoptosis
by camptothecin alone) x 100]. The mean percentage inhibition ± SE is
shown from four experiments.
To better characterize the interaction between curcumin and
camptothecin, studies were pursued with differing exposure times and
concentrations. Inhibition of apoptosis was time dependent, but even
a
relatively brief 3-h incubation inhibited apoptosis by 18.2 ± 2.6%
(Fig. 1D) . Longer incubations of 6 and 9 h resulted in increasing
inhibition of 36.9 ± 6.7 and 56.7 ± 2.4%, respectively, after which a
plateau was reached, during which additional exposure times did not
significantly decrease programmed cell death. Curcumin was able to
inhibit camptothecin-induced apoptosis in a concentration-dependent
fashion, with 1.0, 5.0, and 10.0 µM curcumin inhibiting apoptosis by
9.0 ± 4.7, 43.9 ± 0.9, and 66.3 ± 1.6%, respectively (Table 1) .
Curcumin and Alkylating Agent- and Anthracycline-induced Apoptosis.
Alkylating agents and anthracyclines are commonly applied to the
therapy of patients with breast cancer, and because both classes of
drugs generate ROS (39, 40, 41, 42) and activate JNK (43 , 44) , it
was of interest to determine whether curcumin could inhibit their
ability to induce apoptosis. MCF-7 cells were therefore exposed
either
to mechlorethamine or Adriamycin in culture, and the impact of
curcumin on apoptosis was then evaluated. Mechlorethamine was used as
the alkylating agent rather than cyclophosphamide because, although
the latter is more frequently used clinically, it must be transformed
in vivo to the active metabolite 4-hydroxycyclophosphamide to exert
its effects (45) . Curcumin was able to inhibit the ability of both
mechlorethamine and Adriamycin to induce programmed cell death of
MCF-7 cells in a concentration-dependent fashion (Table 1) . With
respect to Adriamycin, for example, curcumin at 1.0, 5.0, and 10.0 µM
decreased induction of apoptosis by 47.2 ± 6.9, 55.5 ± 4.4, and 65.3
±
6.7%, respectively. To evaluate other model systems, MDA-MB-231 and
BT-474 human breast cancer cell lines were used. In all of the cases
studied, curcumin inhibited the ability of the chemotherapeutic drugs
to activate apoptosis. This effect was dose dependent but even at 1.0
µM, a concentration documented in Phase I chemoprevention trials (25)
, curcumin significantly blocked apoptosis by up to 19.3 ± 2.2 or
27.5
± 16.7%, in the case of BT-474 cells treated with mechlorethamine or
camptothecin, respectively.
Curcumin and Generation of ROS.
ROS are important intermediates in the induction of apoptosis by
chemotherapeutic drugs such as camptothecin and alkylating agents (33
, 37 , 46) . Because curcumin acts as a free radical scavenger, it
was
of interest to evaluate its impact on generation of ROS using
dichlorodihydrofluorescein diacetate. By itself, curcumin had a
minimal impact on ROS in vehicle-treated MCF-7 cells, but treatment
with camptothecin induced a 2.58-fold increase, whereas
mechlorethamine induced a 4.80-fold increase in ROS (Fig. 2A) . When
curcumin was present in conjunction with either of these two
chemotherapeutic agents, it inhibited ROS generation in a
dose-dependent fashion. In the case of mechlorethamine, for example,
1, 5, and 10 µM curcumin inhibited induction of ROS by 35.0, 66.1,
and
72.6%, respectively, compared with the alkylating agent alone. A
similar trend was noted in BT-474 cells in which curcumin inhibited
ROS by 34.7, 48.1, and 56.6%, respectively, (Fig. 2B) at the
concentrations tested. Indeed, 10 µM curcumin decreased ROS in
camptothecin- and mechlorethamine-treated MCF-7 and BT-474 cells to
levels comparable with vehicle-treated cells, demonstrating
curcumin's
ability to act as a free radical scavenger.
Fig. 2. Curcumin inhibits generation of ROS. MCF-7 (A) and BT-474 (B)
cells were either mock treated, vehicle treated, or exposed to
curcumin at 1, 5, and 10 µM. In parallel, they were exposed to either
10 µM camptothecin or 100 µM mechlorethamine, with or without
curcumin, for 12 h. The generation of ROS is assayed using
dichlorodihydrofluorescein diacetate and is expressed as
absorbance/mg
of protein. The mean ± SE is shown from three experiments, each
performed in duplicate. Curcumin and JNK Activation.
ROS have been reported to stimulate SAPK pathways such as JNK (47 ,
48) , and because curcumin inhibits JNK and AP-1 signaling (16 ,
17) ,
we sought to determine its impact on chemotherapy-induced JNK
activation. Using an in vitro immunocomplex assay, curcumin appeared
to have the ability to inhibit JNK activation in a dose-dependent
fashion in MCF-7 cells treated with camptothecin (data not shown). To
characterize this further, AP-1 activity was probed using an ELISA
that detects phospho-c-Jun levels. By itself, curcumin had a mild
impact on AP-1 in vehicle-treated MCF-7 cells, but treatment with
mechlorethamine induced a 1.29-fold increase, whereas camptothecin
induced a 2.63-fold increase in AP-1 activity (Fig. 3A) . When
curcumin was present with either of these two agents, it inhibited
AP-1 activation, and in most cases did so in a dose-dependent
fashion.
In the case of camptothecin, for example, 1, 5, and 10 µM curcumin
inhibited AP-1 activation by 35.0, 39.5, and 44.9%, respectively,
compared with camptothecin alone. A similar trend was noted in
camptothecin-treated BT-474 cells in which curcumin inhibited AP-1
activation by 15.4, 27.7, and 63.7%, respectively (Fig. 3B) , at the
concentrations tested.
Fig. 3. Curcumin inhibits JNK activation. MCF-7 (A) and BT-474 (B)
cells were treated as described in the legend to Fig. 2 , and JNK/AP-
1
activity was evaluated using an ELISA that detects phosphorylated
c-Jun. The mean ± SE is shown from two experiments, each performed in
duplicate, with results expressed in relation to vehicle-only
controls, which were arbitrarily set at 1.00. Curcumin and
Mitochondrial Cytochrome c Release.
Because ROS (reviewed in Ref. 49 ) and JNK activation (50) impact
cytochrome c release from mitochondria, which then activates
apoptosis
(reviewed in 51 ), we postulated that curcumin might be decreasing
mitochondrial cytochrome c release. Thus, the cytosolic fractions
from
cells were isolated, and the presence of cytochrome c was determined
by Western blotting. Both camptothecin and mechlorethamine induced
large amounts of cytochrome c release into the cytosol of MCF-7
cells,
compared with vehicle-treated controls (Fig. 4A and B ,
respectively).
However, curcumin was able, in a dose-dependent fashion, to decrease
the levels of cytosolic cytochrome c. In the case of camptothecin,
for
example, 1, 5, and 10 µM curcumin decreased cytosolic cytochrome c
levels by 40, 58, and 73%, respectively (Fig. 4A) , whereas in
camptothecin-treated BT-474 cells, curcumin decreased cytosolic
cytochrome c by 83, 91, and 93%, respectively (Fig. 4C) . These
studies support the hypothesis that curcumin inhibits topoisomerase 1
inhibitor- and alkylating agent-mediated apoptosis by inhibiting ROS
generation, JNK activation, and mitochondrial cytochrome c release.
Fig. 4. Curcumin inhibits release of cytochrome c into the cytosol.
MCF-7 cells were treated with vehicle and camptothecin (A) or
mechlorethamine (B) either alone or in the presence of curcumin. The
content of cytochrome c in the cytosol was evaluated by Western
blotting, and equal loading was confirmed by reprobing each blot for
heat shock cognate protein 70. Densitometry of the autoradiographs
was
performed for cytochrome c levels, corrected for loading, and the
data
are shown below each panel in relation to the chemotherapy agent
alone, which was arbitrarily set at 1.00. BT-474 cells were similarly
treated in C and D, respectively, and each panel is a representative
result from one of two experiments. V, vehicle; Cam, camptothecin;
Mech, mechlorethamine.
Curcumin and Breast Cancer Xenografts after Cyclophosphamide.
The ability of curcumin to inhibit chemotherapy-mediated apoptosis in
culture suggested that it might have the same activity in vivo. To
evaluate this possibility, xenograft models of human breast cancer
were prepared in nude (nu/nu) mice, and these were randomized to
either a standard diet or one supplemented with curcumin at 25 g/kg
of
feed. This represents 8.7 mM/kg of body weight/day (1 , 2 , and 4 ).
Twenty-four h later, these mice were treated with a single injection
of cyclophosphamide, an alkylating agent that is a component of
several regimens used to treat patients with breast cancer, and the
impact on their tumors was followed daily for 2 days. In pilot
experiments with both MCF-7-based and BT-474-based xenografts, the
animals receiving a regular diet treated with cyclophosphamide had a
significant decrease in tumor size from the day of treatment, or day
1, to day 2. The animals receiving a curcumin-supplemented diet
treated with cyclophosphamide, however, did not have a significant
decrease in tumor size over this time period. Power calculations were
then performed to determine the sample size needed to obtain
statistically significant results, and a second study was performed
using the BT-474 model system. Animals treated with cyclophosphamide
feeding on a standard diet had a significant decrease in their tumor
size from day 1 to day 2, after which the tumors began growing once
again (Fig. 5) . When animals feeding on a curcumin-supplemented diet
were treated with cyclophosphamide, however, their tumors did not
decrease in size and continued to grow from day 1 to day 2 and then
to
day 3 (Fig. 5) . Indeed, whereas tumors in the standard diet group
had
increased by only 1.10 ± 0.18-fold from day 0 to day 2, showing the
effectiveness of day 1 cyclophosphamide therapy, the tumors in the
curcumin diet group had increased by 2.58 ± 0.24-fold compared with
day 0 despite cyclophosphamide treatment (P < 0.0001). In contrast,
control experiments showed no such differences in vehicle-treated
animals (data not shown), with tumors in the standard diet group
increasing by 2.49 ± 0.36-fold from day 0 to day 2, whereas tumors in
the curcumin-supplemented diet increased by 1.97 ± 0.18-fold.
Fig. 5. Dietary curcumin blunts cyclophosphamide-mediated tumor
growth
inhibition. Nude mice bearing BT-474-based xenografts were randomized
on day 0 to receive either standard or curcumin-supplemented diets
and
on day 1 were treated with a single i.p. dose of cyclophosphamide at
265 mg/kg. Tumor weights were followed for a total of 3 days,
calculated using the tumor dimensions, and plotted as the ratio of
the
tumor weight to that on day 0, which is arbitrarily set at 1.00.
Sixteen mice were included in each of these treatment groups, whereas
two additional groups were treated with either a standard diet and
vehicle or the curcumin-supplemented diet and vehicle. These results
are described in the text. Apoptosis and JNK Activation in Xenograft
Tissue.
To determine whether the decreased effectiveness of cyclophosphamide
was attributable to inhibition of programmed cell death, tumor
sections were prepared 24 h after cyclophosphamide. Apoptosis was
then
evaluated by probing for single-stranded sequences after
formamide-induced DNA denaturation (35) , and densitometry was
performed to obtain quantitative results. Comparing the two
cyclophosphamide-treated groups, there was 1.6-fold more apoptosis
detected in the standard diet cohort (Fig. 6A) than the curcumin-diet
cohort (Fig. 6B) . Also, whereas cyclophosphamide increased apoptosis
in the standard diet group compared with treatment with vehicle
alone,
in the curcumin diet group it did not increase the abundance of ssDNA
(data not shown). The activity of the JNK signal transduction pathway
was then evaluated using an antibody that recognizes phosphorylated,
activated SAPK/JNK. Treatment of the standard diet group with
cyclophosphamide resulted in a significant increase in phospho-JNK
reactivity (Fig. 6C) , compared with treatment of the curcumin diet
group (Fig. 6D) . Indeed, this latter group showed no increased
fluorescence density compared with that of the vehicle-treated
curcumin diet group or the vehicle-treated standard diet group. These
studies support the hypothesis that in vivo, as it does in tissue
culture, curcumin can inhibit alkylating agent-induced apoptosis and
JNK activation.
Fig. 6. Dietary curcumin inhibits apoptosis and JNK activation in
vivo. One day after treatment with cyclophosphamide, BT-474-based
tumor xenografts were excised, sectioned, and prepared for
immunofluorescence. Apoptosis induced by cyclophosphamide is shown in
the standard diet group (A) and in the curcumin-supplemented diet
group (B) as red areas containing ssDNA. Phospho-JNK staining is
shown
in C and D for these two groups, respectively, with red areas
indicating presence of phosphorylated, activated JNK. The background
blue staining in all panels is a 4',6-diamidino-2-phenylindole
nuclear
stain. Control slides from vehicle-treated standard diet and curcumin
diet groups were prepared as well. The density of immunofluorescence
staining discussed in the text is the mean from five separate fields
on two duplicate slides.
The chemopreventive activity of curcumin in several animal tumor
model
systems has led investigators to examine its potential impact on
apoptosis with varying results. Some reports suggest that curcumin
inhibits apoptosis such as in human and rat T lymphocytes (30) ,
whereas others have documented an induction of apoptosis in lines
such
as HL60 cells (29) and in vivo in azoxymethane-induced colon tumors
(52) . In some cell lines, there have even been conflicting results
such as in HT-29 human colon cancer cells that have been noted to be
induced into apoptosis by some (53) , whereas others have noted no
effect of curcumin (27) . With respect to cytotoxic chemotherapy,
curcumin has been reported to protect rat lungs and myocardium from
injury by bleomycin (31) and Adriamycin (32) , respectively, and
inhibit apoptosis in UV-irradiated Jurkat cells and
dexamethasone-treated thymocytes (54) , but it could not inhibit
etoposide-induced apoptosis in U937 human monoblastic leukemia cells
(55) . Many of these investigations have used extended incubation
periods with very high curcumin concentrations, however, that would
be
unlikely to be achieved as a result of dietary curcumin intake by
cancer patients. Therefore, we sought to study the effects of
curcumin
using conditions that more closely reproduced those that could be
found in vivo. Because of its ability to act as a free radical
scavenger (6, 7, 8) and to inhibit AP-1 (16) and JNK activation
(17) ,
we hypothesized that because these are important steps in the ability
of some cytotoxic DNA-damaging drugs to induce apoptosis, dietary
curcumin might inhibit the effectiveness of such cancer chemotherapy.
In this study, we demonstrate that curcumin was able, in a dose- and
time-dependent fashion, to inhibit camptothecin-mediated apoptosis in
MCF-7 breast cancer cells (Fig. 1) . This occurred at concentrations
that have been documented in a Phase I chemoprevention trial in
humans, where serum curcumin levels ranging from 0.51 ± 0.11 to 1.77
±
1.87 µM were noted (25) . Serum levels in humans after an oral dose
of
curcumin peak rapidly in as little as 1–2 h, but they decline much
more slowly over the next 12 h (25) , and in our studies, even a
brief
3-h exposure to curcumin was sufficient to significantly inhibit
apoptosis. Moreover, this inhibition was demonstrable in a
concentration-dependent manner using the topoisomerase 1 inhibitor
camptothecin, the alkylating agent mechlorethamine, and the
anthracycline Adriamycin, not only in MCF-7 cells but also in
MDA-MB-231 and BT-474 human breast cancer cells. Such findings
suggest
that this activity is independent of p53 status because MCF-7 cells
are p53 wild type, whereas MDA-MB-231 and BT-474 cells have mutant
p53. Hormone receptor status would also appear to not be a
significant
influence because MCF-7 cells are estrogen receptor positive, whereas
MDA-MB-231 cells are receptor negative (56) . However, additional
experiments, e.g., comparing parental MDA-MB-231 cells and MDA-MB-231
clones that express wild type p53, will be necessary to test these
hypotheses directly.
To evaluate possible mechanisms responsible for this inhibition of
apoptosis, we studied ROS generation and found that curcumin could,
in
a dose-dependent fashion, inhibit the camptothecin- and
mechlorethamine-induced production of ROS (Fig. 2) . JNK activation
with AP-1 activity (Fig. 3) and mitochondrial release of cytochrome c
(Fig. 4) was also inhibited in a concentration-dependent manner.
These
findings support the hypothesis that curcumin inhibits apoptosis by
blocking ROS formation and JNK activation, both of which are
important
signals for cytochrome c release from mitochondria into the
cytoplasm,
which triggers caspase-mediated programmed cell death. If confirmed,
such a mechanism would allow the prediction that drugs that do not
activate JNK should not be influenced by curcumin. Consistent with
this assumption, methotrexate and 5-fluorouracil, two drugs used in
the care of breast cancer patients, which function as antimetabolites
and are not known to activate JNK, were able to induce apoptosis
without any impact by curcumin (data not shown). It should be noted,
however, that Bhaumik et al. (57) have reported that in AK-5 rat
histiocytoma cells, curcumin alone was able to induce apoptosis
through ROS production and cytochrome c release. This difference may
be attributable to the experimental conditions because Bhaumik et al.
used a concentration of 50 µM curcumin and did not study its impact
in
the presence of a chemotherapeutic agent. Alternatively, this may
indicate that there is some cell type specificity to the impact of
curcumin. The current studies also do not rule out a possible
involvement of other pathways that are impacted upon by curcumin in
blocking apoptosis. For example, activation of the transcription
factor NF-B is important in paclitaxel-induced apoptosis (58) , and
because curcumin inhibits NF-B (14 , 15) , this may be another
pathway
involved in its antiapoptotic activity. Additional ongoing studies
using dominant negative mutant constructs that will selectively
inactivate only one pathway at a time will hopefully prove
instructive
in elucidating the molecular basis for this function of curcumin.
Given the ability of curcumin to inhibit apoptosis in tissue culture,
we sought to determine whether it could also do so in vivo, and we
found that dietary supplementation significantly decreased
cyclophosphamide-induced tumor growth delay (Fig. 5) .
Immunofluorescence studies indicated that this occurred in
conjunction
with decreased apoptosis and also with decreased JNK activation (Fig.
6) . This result was somewhat surprising in that, whereas serum
curcumin concentrations in chemoprevention trials have been
comparable
with those used in our in vitro studies, these occurred at a daily
curcumin dose of 8000 mg (25) . Even in populations where dietary
curcumin intake is high, daily exposure is only on the order of 200
mg
(1, 2, 3, 4) . Therefore, it might seem unlikely that such diets
would
result in systemic curcumin levels approaching the conditions we used
in vitro, especially since the oral bioavailability of curcumin is
low
because it is extensively metabolized by the liver. Studies of tumor
sections, however, revealed that those from the two
curcumin-supplemented diet groups showed an immunofluorescence
characteristic of curcumin, whereas the standard diet groups did not
(data not shown; Refs. 1, 2, 3 ). This supports the possibility that,
even after only short-term feeding, curcumin, or one of its
metabolites, can be present in tumor tissue. One possible explanation
for the ability of dietary curcumin to inhibit apoptosis at low
levels
of exposure in vivo is that this may represent a difference in the
absorption and metabolism of curcumin between humans and animals. A
recent study comparing human and rat hepatocytes, however, found that
the major metabolites in both were hexahydrocurcumin and
hexahydrocurcuminol (59) . Alternatively, although curcumin
metabolites have been shown to be less able to inhibit phorbol
ester-induced prostaglandin E2 production in human colonic epithelial
cells than the parental compound (59) , they may be more active than
curcumin in their ability to inhibit ROS formation, JNK activity, and
cytochrome c release. Finally, it is possible that some of the
antiapoptotic effect of curcumin in vivo does not require it to be
present at the site of the tumor. Cyclophosphamide is a prodrug
activated by cytochrome P450s in the liver (45) , and curcumin
inhibits several P450 isoenzymes (18) . The higher concentrations of
curcumin one would expect to find in the liver after a meal
containing
this agent may be sufficient to inhibit conversion of
cyclophosphamide
to its active metabolites, thereby limiting its antitumor efficacy.
Curcumin's ability to inhibit apoptosis in our in vivo studies
suggests that patients with breast cancer who are receiving active
cytotoxic chemotherapy should be excluded from enrollment in any
curcumin-based chemoprevention trials in the future. Because curcumin
has antiangiogenic properties and may have therapeutic potential in
human prostate cancer (24) , our findings also suggest care in its
application to patients with other malignancies receiving concurrent
chemotherapy until this activity of curcumin can be further
investigated. In addition, curcumin is lipophilic and up to 80% is
absorbed by passive diffusion from the intestinal lumen, after which
it concentrates in membranous cellular structures (54) . It is
possible, therefore, that the effects of curcumin might be amplified
in patients with colon cancer receiving chemotherapy unless, as noted
above, it has some cell type specificity, or its antiapoptotic
effects
are mediated through hepatic cytochrome P450. Finally, these findings
strongly support additional research to determine whether breast
cancer, and possibly other cancer patients undergoing chemotherapy,
should limit dietary supplementation of curcumin, which is widely
available over the counter as turmeric extracts, and possibly even
avoid curcumin-containing foods altogether.
FOOTNOTES
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.
1 Supported by the American Institute for Cancer Research Grant
00A097, the Department of Defense Idea and Career Development Award
DAMD17-00-1-0381, and the Leukemia and Lymphoma Society Grant R6206-
02
(to R. Z. O.).
2 To whom requests for reprints should be addressed, at The
University
of North Carolina at Chapel Hill, 22-003 Lineberger Comprehensive
Cancer Center, CB #7295, Mason Farm Road, Chapel Hill, NC 27599-7295.
Phone: (919) 966-9762; Fax: (919) 966-8212; E-mail:
R_Orlowski@....
3 Internet address: http://toxnet.nlm.nih.gov/.
4 The abbreviations used are: NF-B, nuclear factor B; AP-1, activator
protein-1; JNK, c-Jun NH2-terminal kinase; ROS, reactive oxygen
species; LCCC TCF, Lineberger Comprehensive Cancer Center Tissue
Culture Facility; ssDNA, single-stranded DNA; SAPK, stress-activated
protein kinase.
Received 2/15/02; accepted 4/25/02.
REFERENCES
Top
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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If you are Pro-Israel, Jewish,single,21 or over,
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Please also do your part to help Israel by joining pro-Israel groups
both on line and in real life.
Some pro-Israel on line groups are listed below:
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c-span, also known as C-SPAN provides much valuable information.
However, we are
distressed that programs like
Washington Journal on c-span seem to provide a forum for
anti-Semites and enemies of Israel and the Jewish people in
general who seem to not only call into that t.v. show, but get
through, and get
on the air, where they are seldom interupted or cut off by the show's
moderators. We feel that a 2 minute delay could enable the show's
moderators to
screen out the anti-Semites and enemies of Israel and the Jewish
people, however
no such 2 minute delay seems to be in place. Moreover the moderators
of that
show and others do not seem to be of a mind to cut off the anti-
Semites and
other enemies of Israel and the Jewish people in general who
regularly call into
that show and others
on c-span. We have therefore created this Yahoo Group in the hopes
that members
of the group will become regular viewers of c-span and will call in
regularly to
Washington Journal and other c-span call in t.v. shows, to support
Israel and
the Jewish people and to counter the anti-Semites and other enemies
of Israel
and the Jewish people whose calls are regularly aired on c-span. We
also hope
that members of our Yahoo Group will contact their U.S.
Senators and members of Congress and President Bush at
http://www.whitehouse.gov to see what can be done about
the situation at c-span.
http://groups.yahoo.com/group/PeopleForIsrael/
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http://groups.yahoo.com/group/WeAreForIsrael/
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http://groups.yahoo.com/group/WeStandUpForIsrael/
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http://groups.yahoo.com/group/AmericansForIsrael/
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If you know of other pro-Israel on line groups and/or pro-Israel
groups that meet in real life please post the information about them.
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