Post on 13-May-2023
Review Article
Biological Properties of Garlic and Garlic-DerivedOrganosulfur Compounds
Małgorzata Iciek,1 Inga Kwiecien¤ ,2 and Lidia Włodek1*1Chair of Medical Biochemistry, Jagiellonian University,
Medical College, Krakow, Poland2Chair and Department of Pharmaceutical Botany, Jagiellonian University,
Medical College, Krakow, Poland
Medicinal properties of garlic (Allium sativum)have been widely known and used since ancienttimes till the present. Garlic enhances immunefunctions and has antibacterial, antifungal andantivirus activities. It is known to prevent plateletaggregation, and to have hypotensive and choles-terol- and triglyceride-lowering properties,although the latter features have been questioned.This review is focused on anticancer efficacy ofAllium sativum, and attempts to explain the mech-anisms of this action. Medicinal properties of gar-lic rely upon organosulfur compounds mostlyderived from alliin. Organosulfur compounds orig-inating from garlic inhibit carcinogen activation,
boost phase 2 detoxifying processes, cause cellcycle arrest mostly in G2/M phase, stimulate themitochondrial apoptotic pathway, increase acety-lation of histones. Garlic-derived sulfur compoundsinfluence also gap-junctional intercellular communi-cation and participate in the development of multi-drug resistance. This review presents also other lit-tle known aspects of molecular action of garlic-derived compounds, like modulation of cellularredox state, involvement in signal transductionand post-translational modification of proteins bysulfane sulfur or by formation of mixed disulfides(S-thiolation reactions). Environ. Mol. Mutagen.50:247–265, 2009. VVC 2009 Wiley-Liss, Inc.
Keywords: chemoprevention; diallyl polysulfides; agedgarlic extract (AGE); S-thiolation; sulfanesulfur
INTRODUCTION
Garlic (Allium sativum) is one of the oldest medicinal
plants used by different cultures. Already in antiquity it
was used for treatment and prevention of some diseases
[Rivlin, 2001]. The oldest reports of health-promoting
properties of garlic are dated back to the 16th century
BC, when in the so-called Ebers Papyrus from Egypt,
over 20 aliments were purported to be efficiently cured
by garlic [Block, 1985].
The present-day natural medicine recommends to use
garlic to treat parasitoses and other intestinal diseases. Gar-
lic is commonly used in throat infections, digestive tract dis-
orders and fungal infections, like aphthae. Garlic stimulates
the immune system and acts as a natural antibiotic not
harmful for friendly bacterial flora. Many laboratory studies
have confirmed antibacterial, antifungal, antivirus, immu-
nostimulating, and antioxidant properties of garlic [Imai
et al., 1994; O’Gara et al., 2000; Tsao and Yin, 2001;
Corzo-Martinez et al., 2007]. In recent years, garlic has
focused much interest due to its suspected efficacy in cardi-
ovascular diseases since numerous studies have indicated its
cholesterol and triglyceride-lowering, antiaggregatory and
hypotensive potential [Brace, 2002; Gorinstein et al., 2007].
Anticancer properties of garlic were first described by
Weisberger and Pensky in 1958. They reported an inhibi-
tory effect of a garlic extract on cancer cell growth both
in vitro and in vivo [Weisberger and Pensky, 1958]. Since
then intensive laboratory and epidemiological studies
have been carried out to verify chemopreventive and anti-
carcinogenic effects of Allium sativum, and to explain
mechanisms of its action.
Garlic Chemistry
Medicinal properties of garlic and other representatives
of the family Allium (onion, shallot), including their anti-
cancer efficacy, have been attributed to organosulfur com-
pounds.
*Correspondence to: Lidia Włodek, Chair of Medical Biochemistry,
Jagiellonian University, Medical College, Kopernika 7, Krakow 31-034,
Poland. E-mail: mbwlodek@cyf-kr.edu.pl
Received 23 July 2008; provisionally accepted 11 December 2008; and
in final form 11 December 2008
DOI 10.1002/em.20474
Published online 27 February 2009 in Wiley InterScience (www.
interscience.wiley.com).
VVC 2009Wiley-Liss, Inc.
Environmental andMolecular Mutagenesis 50:247^265 (2009)
The most important initial sulfur compound occurring
in the intact garlic bulbs is alliin (S-allylcysteine sulfox-
ide). The whole bulbs contain also g-glutamyl-S-allylcys-
teine (GSAC), S-methylcysteine sulfoxide (methiin), S-
trans-1-propenylcysteine sulfoxide, and S-2-carboxypro-
pylglutathione and S-allylcysteine (SAC), though at much
smaller amounts [Amagase, 2006].
Allicin
Damaging of a garlic bulb by crushing, grinding or cut-
ting induces the release of the vacuolar enzyme alliinase
(alliin: lyase EC. 4.4.1.4) which very quickly, within several
seconds, transforms alliin into allicin via the exceptionally
reactive intermediates, sulfenic acids (R-SOH). Pyruvate
and ammonium ion are by-products of this reaction (Scheme
1). Sulfenic acid, formed in this reaction, undergoes conden-
sation with another sulfenic acid molecule yielding diallyl
thiosulfinate (allicin). The allicin, absent in the intact bulbs,
is the main component of a freshly prepared garlic homoge-
nate [Lanzotti, 2006]. Allicin is poorly soluble in water and
is responsible for the characteristic pungent flavor of garlic.
However, allicin is a very unstable compound. No its traces
were found several minutes after its addition to blood. No
allicin was also detected in urine and blood of people who
used to consume garlic [Freeman and Kodera, 1995]. Com-
mercial preparations of garlic do not contain allicin, either.
Therefore, it seems that considering its exceptional instabil-
ity, allicin cannot be responsible for the biological in vitro
activity of garlic but is an intermediate on the pathway
towards other biologically important sulfur compounds.
Oil-Soluble Organosulfur Compounds
Allicin is easily transformed into oil-soluble polysulfides,
mostly diallyl disulfide (DADS), also into diallyl sulfide
(DAS), diallyl trisulfide (DATS) and diallyl tertasulfide
(Scheme 2). Chemical composition of the preparations
obtained by extraction of oil-soluble garlic fractions
depends on the extraction conditions: temperature, time and
solvent’s polarity. Analysis of allicin solution that had been
allowed to stand at room temperature for 20 hr showed:
66.7% DADS, 14.6% DATS, 13.3% DAS, and 5.4% diallyl
tetrasulfide [Brodnitz et al., 1971]. Some reports suggest
that higher polysulfides, like diallyl penta- heksa- or hepta
sulfides can be formed but their concentrations are low
[O’Gara et al., 2000].
When conditions are appropriate, allicin can be trans-
formed into vinyldithiin and structures of the Z- or E-
ajoene type (Scheme 2). The vinyldithiin was first identified
as a product of thermal degradation of allicin during gas
chromatographic analysis [Brodnitz et al., 1971]. These
structures are formed by dimerization of thioacrolein cre-
ated via allicin b-elimination. Greater amounts of vinyldi-
thiin were produced when a less polar solvent, like hexane,
was used [Iberl et al., 1990]. Ajoene (4,5,9-trithiadodeca-
1,6,11-triene-9-oxide) is generated via allicin S-thiolation
and 2-propenesulfenic acid addition. Ajoene was isolated
from an ether fraction of garlic extract and is a potential
antithrombotic agent [Block and Ahmad, 1984].
Water-Soluble Organosulfur Compounds
The reactions of allicin with ��SH groups can yield
SAC or S-allylmercaptocysteine (SAMC), that are water
soluble compounds [Rabinkov et al., 2000]. Unlike oily
sulfur compounds, water-soluble compounds are odorless
and have more delicate and less characteristic flavor
[Kodera et al., 2002]. These compounds are also formed
during aqueous garlic extraction, when the initial com-
pound GSAC is transformed into SAC and this reaction is
catalyzed by g-glutamyltranspeptidase (gGT) (Scheme 3).
SAC along with its derivatives, S-methylcysteine (SMC)
and SAMC are components of aqueous extracts of garlic
and possess biological activity both in vitro and in vivo.
Garlic Preparations
Garlic essential oil contains only oil-soluble sulfur
compounds (DAS, DADS, vinyldithiins, etc.) without the
water-soluble fraction and allicin. Garlic oil macerate con-
sists of the oil-soluble sulfur compounds and alliin. It
does not contain allicin.
Environmental and Molecular Mutagenesis. DOI 10.1002/em
Scheme1. Conversion of alliin to allicin catalyzed by allinase.
248 Iciek et al.
Garlic powder is generated from garlic cloves which
are crushed and pulverized into powder. It contains alliin
and a small amount of oil-soluble sulfur compounds. Like
all commercial garlic products, it does not contain allicin.
Garlic extracts are produced by soaking of sliced garlic
cloves in extracting solution for a specific time. Then,
after separation of the solution the extract is concentrated.
Out of different garlic extracts, the so-called ‘‘aged garlic
extract’’ (AGE, Kyolic) deserves special attention. AGE
is an odorless product of a prolonged extraction of fresh
garlic at room temperature. Cut or crushed garlic is stored
in 15–20% ethanol solution in water for 20 months.
Then, the extract is filtered and concentrated. Over such a
long time, compounds responsible for characteristic fla-
vor, and pungent and toxic components are naturally
transformed into stable and safe sulfur compounds. AGE
contains most of all water-soluble sulfur compounds
(SAC and SAMC) and small amounts of oil-soluble allyl
sulfides. The main component of Kyolic garlic extract is
SAC, the second largest component is SAMC. These
water-soluble sulfur compounds, formed during garlic
extract aging, have huge antioxidant potential [Corzo-
Martinez et al., 2007]. Comparison of fresh garlic extracts
and AGE in terms of their antioxidant properties indicates
that the latter is more efficient [Harauma and Moriguchi,
2006].
Environmental and Molecular Mutagenesis. DOI 10.1002/em
Scheme 2. Formation of various oil-soluble organosulfur compounds from allicin.
Scheme 3. Formation of water-soluble garlic-derived organosulfur com-
pounds from g-glutamyl-S-allylcysteine. gGT, g-glutamyltranspeptidase.
Anticancer Properties of Garlic 249
Other Components of Garlic
Intact garlic cloves contain also steroidal saponins [Lan-
zotti, 2006] and organic selenium compounds, that possess
a potential anticancer efficacy [Arnault and Auger, 2006].
The main selenium compound is g-glutamyl-Se-methylse-
lenocysteine (GMSC). Like its sulfur analog GSAC, GMSC
can be transformed by gGT to other selenium derivatives,
e.g., Se-methylselenocysteine. Comparative studies of che-
mopreventive efficiency of organic organoselenium com-
pounds and their sulfur analogs demonstrated that diallyl
selenide was 300-fold more effective than DAS in protect-
ing against 7,12-dimethylbenz[a]anthracene-induced mam-
mary adenocarcinomas in rats [El-Bayoumy et al., 2006].
Metabolism of Garlic and Garlic-DerivedOrganosulfur Compounds
Despite a multitude of studies examining biological and
chemical properties of garlic and its organic components,
little is known about metabolism of garlic and its organo-
sulfur compounds in human body.
Studies of Guo et al. conducted on mice demonstrated
that shortly after orally administration of alliin, this com-
pound was observed in the stomach, intestine and liver
without the production of allicin-derived compounds [Guo
et al., 1990]. This allows for conclusion that alliin is not
metabolized to respective organosulfur compounds with-
out an appropriate enzyme (allinase).
Allicin added to fresh blood is quickly transformed into
allyl mercaptan but this compound was not found in
blood or urine of people who used to consume garlic,
what suggests that it is further transformed [Lawson and
Wang, 2005]. GC-MS analysis reported by Minami et al.
showed a content of allyl mercaptan and DADS in air
exhaled by people consuming garlic [Minami et al.,
1989]. On the other hand, Rosen et al. demonstrated that
allyl methyl sulfide (AMS) was the main volatile metabo-
lite in the exhaled air after garlic consumption; DAS and
DADS were detected at lower quantities [Rosen et al.,
2000]. These seemingly contrasting findings are probably
attributable to the fact that the analyses were conducted at
different times after consuming garlic. It seems that allyl
mercaptan created during allicin metabolism is an inter-
mediate formed immediately after garlic consumption.
With time elapsing, it is methylated in the reaction with
S-adenosylmethionine (SAM) yielding AMS (Scheme 4).
Hence, AMS is the main metabolite of allicin in raw garlic,
while allyl mercaptan is an intermediate. The studies of
Lawson convincingly demonstrated that the level of AMS
in the exhaled air depended on amount of the ingested alli-
cin or its derivatives [Lawson and Wang, 2005].
Allyl mercaptan was not detected in the exhaled air of
people who had eaten cooked whole garlic cloves. It is
not surprising because cooking completely inactivates alli-
nase necessary for alliin transformation into allicin. There
are reports based on in vitro studies that some intestinal
bacteria express allinase activity [Knobloch, 2000]. If it is
so, alliin present in the cooked garlic cloves could be
transformed by bacterial enzymes into biologically active
compounds. However, Lawson’s analysis of air exhaled
after consuming cooked garlic did not confirm this hy-
pothesis [Lawson and Wang, 2005].
Studies of Germain et al. on an animal model after
DADS treatment demonstrated in urine the presence of
oxidized forms of AMS: allyl methyl sulfoxide (AMSO)
and allyl methyl sulfone (AMSO2) [Germain et al., 2002].
Environmental and Molecular Mutagenesis. DOI 10.1002/em
Scheme 4. Proposed scheme for the metabolism of DADS, DATS and
allicin (A) and DAS transformations (B). SAM, S-adenosylmethionine;
SAH, S-adenosylhomocysteine; CYP2E1, isoenzyme of cytochrome P450.
250 Iciek et al.
This suggests that AMS, the main garlic metabolite of
allicin or its derivatives can undergo further transforma-
tions, as depicted in Scheme 4.
However, AMS was not detected in exhaled air after
ingestion of DAS and vinyldithiin, which suggests that
they are metabolized via different still unknown routes
[Lawson and Wang, 1993]. On the other hand, Davenport
suggested that DAS could be metabolized by one of the
cytochrom P450 isoenzymes to form diallyl sulfoxide
(DASO) and then diallyl sulfone (DASO2) [Davenport
and Wargovich, 2005] (Scheme 4).
Studies of the metabolism of a water-soluble compound
SAC indicated that SAC was detected in blood and its
concentration and other pharmacokinetic parameters were
correlated with SAC doses administered to animals
[Nagae et al., 1994]. Urine of animals orally administered
SAC was shown to contain marked amounts of N-acetyl-
S-allylcysteine, which suggests that SAC can be trans-
formed in vivo by N-acetyltransferase into the N-acety-
lated metabolite [Jandke and Spiteller, 1987]. SAC was
found also in human blood after ingesting of AGE, the
main component of which is SAC (Steiner and Li, 2001).
As can be seen from the above overview, the garlic
metabolism still is not fully understood. Most studies
focused on the exhaled air but it seems that a reliable
analysis of relevant blood components at different inter-
vals after garlic consumption and its organosulfur com-
pounds would be more informative.
Garlic for Cancer PreventionçEpidemiological Studies
Numerous studies have suggested that garlic possesses
anticancer activity. Epidemiological studies were con-
ducted in human populations greatly differing in con-
sumption of plants of the family Allium. Over the last
30 years, there have been many literature reports of epi-
demiological studies which have indicated that a garlic-
rich diet decreases risk of some cancers. Several of these
studies are described below.
One of earlier trials carried out in China compared two
big human populations living in regions differing in garlic
consumption. Mortality in stomach cancer patients from the
region where people have consumed high-garlic diet (about
20 g a day) was three times lower than in the second region
in which consumption of plats of the family Allium was
very low (less than 1 g a day) [Mei et al., 1982]. The
authors postulated that garlic inhibited reduction of nitrates
to nitrites with bacterial participation, which lowered nitrite
concentration in gastric juice, thereby decreasing produc-
tion of carcinogenic nitrosoamines.
Garlic consumption is also negatively correlated with co-
lon cancer as reported by Steinmetz et al. [1994]. Epidemi-
ological analysis of Fleischauer et al., who investigated the
dependence between garlic consumption and incidence of
gastric and colon cancer revealed that the consumption of
larger amounts of raw garlic correlated with a lower risk of
these types of cancer. The consumption of cooked garlic
had no effect [Fleischauer et al., 2000].
Chinese studies examined the relationship between the
consumption of Allium vegetables and prostate cancer risk.
These studies comprised about 450 control men and 250
prostate cancer patients. It was shown that the consumption
of large amounts of garlic and other vegetables of this fam-
ily (above 10 g daily) significantly reduced risk of prostate
cancer [Hsing et al., 2002]. The notable feature of this study
was that the reduced risk of prostate cancer was independ-
ent of various others factors such as consumption of other
foods or body weight. These reports were confirmed by an
independent research of Key et al. [1997].
On the other hand, French studies confirmed a reduced
risk of breast cancer with increased consumption of garlic
and onion [Challier et al., 1998]. Earlier studies con-
ducted in Greece and in the Netherlands showed no effect
of Allium sativum on the risk of breast cancer [Katsouanni
et al., 1986; Dorant et al., 1995]. These discrepant data
question reliability of these studies and preclude unequiv-
ocal conclusions as to a correlation between garlic con-
sumption and breast cancer risk.
Epidemiological studies of this type are not a fully reli-
able source of information since in a majority of cases
they are based on interviewing the patients about the
amount of garlic or other plants of the family Allium in
their diet. For this reason, these studies are a kind of
approximation because it is difficult to precisely define a
daily or weekly garlic consumption by study participants,
particularly over many years. Moreover, often these stud-
ies do not account for other factors (genetic or environ-
mental, smoking and alcohol consumption) distinguishing
study populations and undoubtedly influencing the risk of
cancer. Frequently, it is not specified what form of garlic
was administered (raw or cooked). It is commonly known
that many other dietary components, like green tea or
green vegetables and some fruits also significantly influ-
ence cancer risk. However, the interviews on which the
discussed studies were based contained only data regard-
ing plants of the family Allium, and did not analyze all
potentially anticancer dietary components.
A double-blind intervention study performed in China
showed that the high dose of DATS in combination with
microdoses of selenium offered protection against gastric
cancer [Li et al., 2004]. These studies comprised 2,526 sub-
jects in the intervention group and 2,507 subjects in the
control group. Supplements (DATS in a dose of 200 mg per
day and selenium 100 lg per day) were taken by the inter-
vention group for 1 month per year during 3 years. Control
group was given placebo for the same time period. Interest-
ingly, the authors observed protective effects against gastric
cancer for males but not for females. Although these studies
analyzed the influence of other factors like age, smoking,
alcohol consumption and medical history of stomach ill-
Environmental and Molecular Mutagenesis. DOI 10.1002/em
Anticancer Properties of Garlic 251
ness, but the phenomen of sex dependence was not dis-
cussed in detail in that article. Moreover, other dietary com-
ponents with potential anticancer properties were not taken
into account. Whether supplementation of even relatively
high doses of DATS only for 1 month per year can signifi-
cantly influence incidence of cancer within 5 years after
supplementation, also rises some doubts.
Nonetheless, most of these studies suggest that the diet
rich in garlic or other Allium plants decreases the risk of
some cancers, particularly digestive tract cancers. Even if
this effect is attributable not only to garlic, it seems
advisable not to forget about garlic and other Allium plants
in regular diet, the more so that no studies have indicated an
increase in cancer risk with increased garlic consumption.
Anticancer Effect of Garlic and Garlic-Derived Compoundsin Animal Studies
Studies on animals models found in literature were car-
ried out using either individual garlic-derived compounds
or fresh garlic macerate. Belman was the first to describe
chemopreventive effects of garlic oil against skin tumori-
genesis initiated by 7,12-dimethyl benz(a)anthracene
[Belman, 1983]. Development of the aflatoxin B1- or
diethylnitrosamine-induced liver cancer in rats was effi-
ciently limited by fresh garlic [Samaranayake et al., 2000]
and garlic oil [Soni et al., 1997].
Individual Allium-derived OSCs, mostly oil-soluble, are
highly effective in suppressing cancers induced by certain
chemical carcinogens in animal models. For example, car-
cinogenesis induced by benzo[a]pyrene in the mouse for-
estomach was significantly inhibited by DAS and DATS
[Sparnins et al., 1988]. Administration of DADS afforded
protection against colon and renal neoplasia in a multior-
gan carcinogenesis model in rats [Takahashi et al., 1992].
Cancer chemoprevention by DAS or DADS has also been
observed against N-nitrosomethylbenzylamine-induced
esophageal cancer and dimethylhydrazine-induced colon
cancer in rats [Wargovich et al., 1988], as well as N-
methyl-N-nitrosourea and 2-amino-1-methyl-6-phenylimi-
dazol[4,5-b]pyridine-induced mammary cancer in rats
[Schaffer et al., 1996; Suzui et al., 1997].
Moreover, garlic components have been shown to in-
hibit cancer cell growth in vivo in the xenograft models,
what was presented by Sundaram and Milner (1996b).
They have reported an inhibitory effect of DADS on
growth of human colon tumor cells (HCT-15) implanted
in nude mice. Studies from the laboratory of Singh et al.
have shown that DADS suppressed growth of H-ras onco-
gene-transformed tumor xenografts in nude mice without
any side effects [Singh et al, 1996]. Similarly, DATS sig-
nificantly inhibited growth of PC-3 human prostate cancer
xenografts in male nude mice [Xiao et al., 2006].
All above-presented studies carried out on animal mod-
els suggest that garlic oil and oil-soluble organosulfur
compounds (most of all allyl sulfides) are effective in
affording protection against some types of cancer, mostly
gastrointestinal, induced by a variety of chemical carci-
nogens. The compounds under study were most often
administered by oral intubation. Such in vivo experiments
the most closely resemble physiological conditions, which
makes them a reliable source of information about biolog-
ical activity of garlic-derived compounds. Hence, numer-
ous studies have confirmed that allyl sulfides inhibit
development of some cancers, although Belman’s studies
indicated that onion oil was a more potent inhibitor of
tumor promotion [Belman, 1983].
Concerning in vivo studies, it may be doubtful whether
all doses of allyl sulfides used in literature, apart from posi-
tive effects inhibiting cancer growth, indeed did not cause
adverse side effects on normal cells. Some studies have
suggested that normal cells were more resistant to apoptosis
induction by OSCs compared with cancer cells [Karmakar
et al., 2007; Kim et al., 2007; ], however, the mechanism of
this selectivity remains unknown. On the other hand, other
studies have demonstrated toxicity of higher doses of fresh
garlic extract in normal cells of the gastrointestinal tract
[Nakagawa et al., 1980; Joseph et al., 1989]. Our unpub-
lished studies on Swiss mice also suggested toxicity of
higher doses of DATS (62 mg per kg of body weight) caus-
ing death of animals. Analogous doses of DAS and DADS
did not induce such toxic effects. These facts question
safety and selectivity of the use of garlic-derived allyl sul-
fides, particularly DATS. Therefore, as well efficacy as
safety of application of garlic-derived OSCs depends on
proper dose, which remains to be established in future.
Mechanisms of Anticancer Action of Garlic andGarlic-Derived Organosulfur Compounds
Studies of recent years have focused on elucidation of
the mechanism of biological activity of garlic. Hundreds
studies were conducted both in vivo and in vitro using
individual organic sulfur compounds, mostly allyl sulfides
and their metabolites or water-soluble compounds (SAC,
SAMC) [Thomson and Ali, 2003; Herman-Antosiewicz
and Singh, 2004; Herman-Antosiewicz et al., 2007].
Although the precise mechanism of anticancer efficacy of
garlic is still unknown, several hypotheses were presented
based on experimental studies: antioxidant action, inhi-
bition of carcinogen activation, enhancement of phase 2
detoxification, induction of apoptosis, cell cycle arrest,
modulation of cellular redox status and signal transduc-
tion, post-translational modification of proteins by forma-
tion of mixed disulfides, hydropersulfides and trisulfides.
Antioxidant Effects of Garlic and Aged Garlic Extract
Cancer, like many other human pathologies (e.g., cardi-
ovascular, neurodegenerative and inflammatory diseases)
Environmental and Molecular Mutagenesis. DOI 10.1002/em
252 Iciek et al.
is connected with oxidative modifications of biological
molecules, mainly, proteins, lipids and nucleic acids by
reactive oxygen species, among them free radicals. To
protect cellular macromolecules against toxic free radical
and nonradical oxidants, cells have developed antioxidant
defense systems that includes antioxidant enzymes, such
as superoxide dismutase (SOD), catalase, glutathione per-
oxidase (GP) and low-molecular-weight antioxidants
including glutathione (GSH).
It has been reported that permanent garlic consumption
significantly increases the antioxidant activity of cells
[Banerjee et al., 2002]. Among garlic-derived products,
AGE is the preparation with even higher antioxidant
activity than fresh garlic and other commercial garlic sup-
plements [Borek, 2001]. Imai and coworkers studied the
antioxidant properties of three garlic preparations and
organosulfur compounds derived from garlic in the liver
microsomal fraction. They found that major organosulfur
compounds from AGE, SAC and SAMC exhibited a
potent radical scavenging activity [Imai et al., 1994].
Other nonsulfur components of AGE; N-fructosyl-arginine
and N-fructosyl-glutamate were shown to have antioxidant
activity, which was comparable to that of ascorbic acid
[Ryu et al., 2001]. AGE exerts antioxidant action not only
by scavenging reactive oxygen species (ROS) but also by
enhancing the cellular antioxidant enzyme activities and
by increasing glutathione level in the cells. AGE inhibits
lipid peroxidation and oxidation of low density lipoprotein
(LDL) thereby decreasing the risk of cardiovascular dis-
eases [Lau, 2006]. Balasenthil and coworkers have inves-
tigated the effect of aqueous extract of garlic on lipid per-
oxidation and levels of antioxidants during buccal pouch
carcinogenesis in hamsters. They observed that the admin-
istration of AGE diminished lipid peroxidation in oral
tumor tissue and increased GSH level and glutathione per-
oxidase activity [Balasenthil et al., 1999]. AGE inhibited
LDL oxidation and minimized endothelial cell injury by
preventing depletion of intracellular GSH and by remov-
ing peroxides [Ide and Lau, 1999]. Borek in her review
has suggested that AGE protects endothelial cells from
ROS-induced injury by modifying cellular scavenging
enzymes, among other things [Borek, 2001]. AGE in a
dose- and time-dependent fashion suppressed the produc-
tion of hydrogen peroxide and superoxide radical, what
was accompanied by an increase in SOD, catalase and
glutathione peroxidase activity [Wei and Lau, 1998].
AGE also exerts an anti-inflammatory effect by inhi-
biting the oxidative stress-induced activation of nuclear
factor kappa B (NFjB), which is implicated in the expres-
sion of pro-inflammatory enzymes, such as inducible
nitric oxide synthase and cyclooxygenase-II. NFjB acti-
vation by reactive oxygen species has been also impli-
cated in the regulation of gene transcription. Some studies
have demonstrated that SAC inhibited hydrogen peroxide
or tumor necrosis factor a (TNFa)-induced NFjB activa-
tion in endothelial cells [Ide and Lau, 2001]. Moreover,
garlic extract exerted radioprotective effects, protecting
the cells against ionizing radiation and UV-induced dam-
age [Chittezhath and Kuttan, 2006].
Banerjee et al. in their review confirmed that various
preparations of garlic, mainly AGE have been shown to
possess a promising antioxidant potential, however, they
noted that raw garlic homogenate at higher doses has
been shown to be toxic to the liver and other tissues
[Banerjee et al., 2003]. Indeed, apart from antioxidant
effects of AGE and some others garlic compounds, there
are a few reports highlighting the toxic effects of garlic.
Nakagawa reported that raw garlic juice at a high dose
(5 ml/kg of body weight) led to death of rats due to stom-
ach injury [Nakagawa et al., 1980]. Another study demon-
strated that the administration of aqueous raw garlic
extract in drinking water significantly elevated aspartate
aminotransferase level, suggesting liver injury [Joseph
et al., 1989]. Moreover, the administration of garlic oil
significantly reduced the body weight gain of rats, sug-
gesting a toxic effect [Sheen et al., 1999]. Banerjee et al.
compared these toxic effects of garlic extracts to the same
proooxidant effects of some other known antioxidants
used at high concentrations [Banerjee et al., 2003]. Vita-
min C in excess may act as a prooxidant in the presence
of the transition metals Fe31 or Cu21 and cause lipid per-
oxidation [Podmore et al., 1998; Halliwell, 2000]. Beta-
carotene given at high doses to smokers has been shown
to have harmful effects in clinical studies because of its
prooxidant actions [Siems et al., 2005].
In the light of the aforementioned reports about toxic
effect of high doses of raw garlic or its oil-soluble deriva-
tives, it seems that the commonly accepted hypothesis
about antioxidant action of garlic is controversial. Data on
antioxidant properties of AGE are much more convincing.
Therefore, while looking for an efficient preparation to
lower the level of reactive oxygen species and lipid per-
oxidation, it is better to use Kyolic extract than to con-
sume larger amounts of fresh garlic.
Effect of Organosulfur Compounds on Detoxificationof Xenobiotics
The process of detoxification of xenobiotics is known
to be comprised of two phases. Phase 1 involves microso-
mal transformation of xenobiotic molecules by cyto-
chrome P450. This phase catalyzed by monooxygenases
(Cyt P450) consists mostly of the reactions of hydroxyla-
tion, oxidation or hydrolysis yielding modified derivatives.
In phase 2 these derivatives are conjugated with glucu-
ronic acid, glutathione or sulfate. The aim of both phases
of metabolism of drugs and other xenobiotics is to
increase their polarity and solubility, to lower their toxic-
ity and to facilitate their excretion. Paradoxically, some-
times phase 1 reactions catalyzed by cytochrome P450
Environmental and Molecular Mutagenesis. DOI 10.1002/em
Anticancer Properties of Garlic 253
produce more chemically reactive, toxic and carcinogenic
compounds than parent compounds. Some most powerful
carcinogens are formed in vivo from harmless compounds
via monooxygenase-catalyzed reactions.
Organosulfur compounds (OSCs) derived from garlic
can inhibit experimental cancer in various animal models
through modification of carcinogen-detoxifying enzymes,
such as cytochrome P450 [Chun et al., 2001]. Both DAS
and DADS efficiently inhibit one of isoenzymes of cyto-
chrome P450 CYP2E1, which is responsible for the acti-
vation of nitrosoamines, hydrazines and benzene [Wargo-
vich, 2006]. CYP2E1 inhibition decreases carcinogenic
properties of these compounds.
Yang et al. investigated the effect of DAS and its oxida-
tion products diallyl sulfoxide (DASO) and diallyl sulfone
(DASO2) on chemical carcinogenesis and mutagenesis
[Yang et al., 2001]. All these compounds limited develop-
ment of chemically induced cancers by blocking the phase 1
enzymes. The protective effect was observed when organu-
sulfur compounds were administrated before, during or soon
after treatments with some chemicals: carbon tetrachloride,
N-nitrosodimethylamine and acetaminophen in rodents.
DAS, DASO and DASO2 inhibit competitively CYP2E1, a
major carcinogen-activating enzyme. DAS oxidation to
DASO and DASO2 is catalyzed by cytochrome P450, there-
fore, as they are the substrates of CYP2E1 they compete for
its binding site and limit ability of the enzyme to activate
other carcinogenic substrates [Brady et al., 1991].
Davenport and Wargovich evidenced that organosulfur
compounds derived from garlic (DAS, DADS and AMS)
significantly decreased hepatic CYP2E1, which correlated
with a diminished p-nitrophenol hydroxylase (PNPH)
activity [Davenport and Wargovich, 2005]. On the other
hand, none of the tested OSCs modulated hepatic CYP2E1
mRNA level. The authors have suggested that CYP2E1
activity is regulated by garlic-derived compounds via post-
translational modification. In fact, DAS can be metabo-
lized by CYP2E1 by oxidation at the sulfur atom to form
DASO and then DASO2. The final product of this oxida-
tion, an epoxide can bind to the enzyme and cause its
inactivation. Furthermore, these authors demonstrated that
apart from CYP2E1 inhibition, DAS and AMS increased
hepatic level of CYP1A family enzymes without signifi-
cantly increasing their mRNA [Davenport and Wargovich,
2005]. These data are in agreement with reports of Guyon-
neta et al, who tested in vivo the effect of some OSCs on
the modification of some CYP izoenzymes and the activa-
tion of various carcinogens [Guyonnet et al., 2000]. In that
study, DAS, dipropyl sulfide (DPS) and dipropyl disulfide
(DPDS) slightly increased ethoxyresorufin O-deethylase
(EROD) and methoxyresorufin O-demethylase (MROD)
activities of CYP1A family and strongly enhanced pentox-
yresorufin O-dealkylase (PROD) activity.
Similar studies conducted by Wu et al. evidenced that
the levels of cytochrome P450 1A1, 2B1, and 3A1 were
higher in OSC-treated group than in the control. It was
accompanied by elevated activities of EROD and PROD.
In contrast, expression of P450 2E1 protein was signifi-
cantly suppressed by all tested allyl sufides (DAS, DADS,
DATS) [Wu et al., 2002].
Induction and stabilization of CYP1A enzymes by garlic
OSCs may potentiate tumor development, since these
enzymes are involved in the activation of several potential
human carcinogens. However, some studies have sug-
gested that CYP1A enzymes may fulfill a protective role
against potentially toxic compounds [Gonzales and
Kimura, 2003]. Hence, stabilization of these enzymes may
prevent binding and subsequent metabolic activation of
procarcinogens, whereas their induction may increase the
clearance rate of toxic metabolites.
There are numerous evidences that garlic-derived or-
ganic sulfur compounds induce phase 2 enzymes, i.e., glu-
tathione S-transferase, epoxide hydrolase, quinone reduc-
tase and glucuronate transferase, which also increases the
clearance rate of toxic compounds. Glutatione S-transfer-
ases (GST) are important detoxifying enzymes that remove
harmful electrophiles, including carcinogens, by conjugat-
ing them with glutathione. Any substance that increases
the levels or activity of GSTs has a potential to be chemo-
preventive. Many authors have described the elevation
of glutathione S-transferase activity by DAS and DADS
administered orally or intraperitoneally [Sheen et al.,
1999; Guyonnet et al., 2001]. Fukao et al. found that ipadministration of DATS (10 lmol/kg of body weigh) to
rats caused a marked increase in the activities of gluta-
tione-S-transferase (GST) and quinone reductase (QR)
activity, whereas the same doses of DAS and DADS
increased the activity of these enzymes only slightly
[Fukao et al., 2004]. Our unpublished studies conducted
on mice indicated that DAS, DADS at higher doses (350
lmol/kg) and DATS (120 lmol/kg) significantly increased
the GST activity. More recently, Tsai et al. have reported
that DADS and DATS up-regulated the gene expression of
the p class of glutathione S-transferase in clone 9 cells
[Tsai et al., 2007]. Chen et al. investigated the transcrip-
tional levels of NAD(P)H: quinine oxidoreductase 1
(NQO1) and heme oxygenase 1 (HO1) genes after adminis-
tration of three major garlic OSCs: DAS, DADS and DATS
in human hepatoma HepG2 cells. NQO1 gene expression
was elevated by all three tested compounds, and DATS eli-
cited the strongest inductive effect among them. Similarly,
HO 1 gene expression was increased by treatments with
DADS and DATS, but not DAS [Chen et al., 2004].
Many studies have proven that allyl-containing OSCs
(DAS, DADS, DATS) are more potent in affecting detoxi-
fying enzymes than the propyl-containing OSCs (DPS,
DPDS) [Chen et al., 2004]. Wargovich has suggested that
allyl group-containing DAS and AMS are the strongest
inhibitors of the CYP2E1 protein [Wargovich, 2006].
Moreover, the potency in modulating the activity and
Environmental and Molecular Mutagenesis. DOI 10.1002/em
254 Iciek et al.
expression of the enzymes involved in detoxification sys-
tems is often correlated with the number of sulfur atoms in
allyl sulfides. It seems that monosulfides (e.g., DAS) have
greater effect on cytochrome P450 than analogical di- lub
trisulfides. Wu et al. reported that the CYP1A1, 2B1, and
3A1 protein levels were increased by allyl sulfides with the
following order of potency: DAS > DADS > DATS [Wu
et al., 2002]. On the contrary, DADS and DATS showed
greater potency than DAS (DATS > DADS > DAS) in
affecting the activity of phase 2 enzymes, mainly GST.
Based on these studies, it can be concluded that oil-
soluble organosulfur compounds significantly affect both
phase 1 and phase 2 metabolism of xenobiotics, which
can directly influence carcinogen activation.
Cell Cycle Arrest
Eukaryotic cell cycle consists of S phase (DNA replica-
tion) and M phase (nucleus and cytoplasm division).
These key events are spaced by intervals of growth and
reorganization: G1 and G2 phase. Cell cycle progression
requires the regulation of different cyclins, cyclin-depend-
ent kinases (Cdk) and Cdk inhibitors. Another level of
control relies on reversible phosphorylation of several reg-
ulatory proteins [Morgan, 1995; Murray, 2004]. Progres-
sion of cells from G2 to M phase requires activation of
Cdk1/cyclin B kinase complex. Its activity is regulated by
two mechanisms: (1) association with regulatory cyclin B
and (2) phosphorylation and dephosphorylation.
Cell cycle arrest occurs in response to cellular stress
through activation of some signal transduction pathways
(checkpoints) [Hartwell and Weinert, 1989]. These check-
points are activated in the G1/S phase to prevent replica-
tion of damaged DNA or in the G2/M phase to prevent
segregation of damaged chromosomes during mitosis.
Many medicines or dietary components showed antiproli-
ferative effects through blocking cells within the G1/S or
G2/M phase of the cell cycle [Wu et al., 2004]. Many
studies have shown that treatment of various cancer cells
with garlic organosulfur compounds leads to cell cycle
arrest [Knowles and Milner, 2000a, 2001].
Studies of Knowles and Milner revealed that antyproli-
ferative effects of DADS in cultured human colon tumor
cells (HCT-15) were related to its ability to increase the
number of cells in the G2/M phase that was accompanied
by a decrease in the number of cells in G1 and S phase
[Knowles and Milner, 1998]. Moreover, DADS exposure
inhibited Cdk1 (also known as p34cdc2) kinase activity.
In addition, those studies revealed that the increased pro-
portion of HCT-15 cells in the G2/M phase following
DADS treatment was accompanied by an increase in cyclin
B1 protein expression [Knowles and Milner, 2000b].
Other studies of those authors demonstrated that the sup-
pression of p34cdc2 kinase activity by DADS did not
results from directs interactions with the protein, but from
the changes in factors influencing the formation and con-
version of the enzyme to its active form, such as a reduc-
tion in the Cdk1-cyclin B1 complex formation, inactivat-
ing Cdk1 phosphorylation, and decrease in Cdc25C phos-
phatase level [Knowles and Milner, 2000b].
Wu and coworkers investigated the effect of various
allyl sulfides (DAS, DADS, and DATS) on cell cycle reg-
ulation in human liver tumor cells J5. They found that J5
cells were significantly arrested in G2/M phase by DADS
and DATS treatments, and DATS was more effective
than DADS. These authors suggested that DATS could
arrest the J5 cells in G2/M phase of cell cycle by increas-
ing of cyclin B1 and decreasing of Cdk7 kinase expres-
sion [Wu et al., 2004]. Cell cycle arrest in the G2/M
phase upon treatment with DADS has also been reported
in other tumor cell lines. Gunadharini et al. studied anti-
proliferative effects of DADS on prostate cancer cells in
vitro. They reported that DADS (in a dose-dependent
manner) suppressed growth of LNCaP cells [Gunadharini
et al., 2006] and induced cell cycle arrest in G2/M transi-
tion in PC3 cells. This inhibition was accompanied by
down-regulation of Cdk1 (p34cdc) with an increase in
cyclin B1 expression [Arunkumar et al., 2006]. Xiao and
Singh studied the effects of DAS, DADS and DATS on
cell cycle progression using PC-3 human prostate cancer
cells. The results showed that DADS and DATS inhibited
proliferation of PC-3 cells in a dose-dependent manner,
whereas DAS was minimally active. This inhibition (G2/
M phase arrest of PC-3 cells) was associated with a sig-
nificant reduction in the level of Cdc25C phosphatase
[Xiao and Singh, 2003]. Other studies of these authors
conducted on human prostate cancer cells PC-3 and
DU145 and in parallel on normal prostate epithelial cell
line PrEC revealed that the treatment of these cells with
DATS caused enrichment of the G2/M fraction in PC-3
and DU145 cells, but not in the normal epithelial cells.
The authors suggest that DATS-induced G2/M phase cell
cycle arrest in human prostate cancer cells is connected
with hyperphosphorylation and reactive oxygen species-
mediated destruction of Cdc25C [Xiao et al., 2005a].
Other studies of Xiao et al. conducted on the colon
cancer cells SW480 revealed that among various tested
compounds (e.g., SAC, SAMC, DAS, DADS and syn-
thetic compound S-trityl-L-cysteine) only SAMC, DADS
and trityl-cys affected cell cycle progression and caused
cell cycle arrest in the G2/M phase. They found that the
treatment of cells with SAMC or trityl-cys induced a dra-
matic increase in the number of cells in M phase and
only a slight increase in cells in the G2 phase. It indicates
that these compounds arrested cells in mitosis. However,
DADS at the tested concentrations inhibited both the G2
and the M phase progression [Xiao et al., 2005b]. The
authors suggested that both the allyl and disulfide moi-
eties were important features for antiproliferative effects
of garlic compounds.
Environmental and Molecular Mutagenesis. DOI 10.1002/em
Anticancer Properties of Garlic 255
Yuan and coworkers investigated the role of p38 MAP
kinase signal transduction pathways in DADS-induced
G2/M arrest in human gastric cancer MGC803 cells. p38
MAP kinase (p38) is a member of the mitogen-activated
protein (MAP) kinase signaling cascade, which has been
shown to regulate a variety of cellular events such as cell
proliferation, apoptosis and differentiation. They found
that DADS induced growth inhibition of MGC803 cells
and arrest in the G2/M phase. This event involved activa-
tion of p38 MAP kinase pathways. Decreased Cdc25C
protein expression by p38 played a critical role in G2/M
arrest by DADS, but other mechanisms cannot be
excluded [Yuan et al., 2004].
Histone Acetylation
In eukaryotic cells, the nuclear DNA is tightly wrapped
around core histone proteins, which are organized in octa-
mers composed of two dimmers H2A- H2B and tetramer
(H3-H4)2. All core histones can be reversibly modified by
acetylation, methylation, phosphorylation, ubiquitination
and biotinylation [Espino et al., 2005]. Acetylation is one
of these post-translational histone’s modifications which
seems to be an important mechanism for the regulation of
gene transcription. Acetylation of specific histone lysine
residues within the N-terminal domain of the core histo-
nes reducing interactions between DNA and histones may
partially activate gene expression through an increased
accessibility of DNA to transcription factors [Legube and
Trouche, 2003].
Garlic-derived OSCs have been shown to increase acet-
ylation of the core nucleosomal histones in various cell
lines in vitro. Increased acetylation of histones might
result from augmented activity of histone acetyltransfer-
ases or decreased histone deacetylase (HDAC) activity.
Lea et al. reported the increased acetylation of H3 and H4
histones in DS19 mouse erythroleukemic cells and in K562
human leukemic cells after treatment of some of OSCc
[Lea et al., 2002]. Acetylation was also induced in rat
hepatoma and human breast cancer cells by DADS and its
metabolite, allyl mercaptan. This increase in histone acet-
ylation was associated with the inhibition of HDAC activ-
ity, and AMS was shown to be a more potent inhibitor of
this enzyme’s activity than DADS [Lea et al., 1999]. The
induction of histone acetylation was also observed by
these authors in Coco-2 human colon cancer and T47D
human breast cancer cells upon the treatment with water-
soluble garlic compounds, SAMC and S-allyl cysteine
(SAC). SAMC was a much more potent inducer of histone
acetylation than SAC, but no significant effects of these
compounds on histone deacetylase activity was observed
in that study [Lea et al., 2002]. The same authors have
investigated the effect of DADS and AMS on histone
acetylation in tumor-bearing rats. They observed an
increased acetylation of histones in the liver and Morris
hepatoma 7,777 cells in vivo [Lea and Randolph, 2001].
Druesne et al. have shown that DADS-induced acetyla-
tion of histones is connected not only with the inhibition
of histone deacetylase activity but also with hyperacetyla-
tion of histones H3 and H4 and induction of Cdk1 inhibitor
p21. HDAC inhibitors can activate expression of several
genes, including p21waf1/cip1 gene, which is a down-regu-
lating protein of the cell cycle progression. These authors
revealed that DADS induced an increase in p21waf1/cip1
expression at mRNA and protein levels in two human
colon adenocarcinoma Caco-2 and HT-29 cell lines.
These results suggest that DADS could inhibit cell prolif-
eration through the inhibition of HDAC activity, histone
hyperacetylation and increase in p21waf1/cip1 expression
[Druesne et al., 2004]. Besides, these authors investigated
for the first time the effects of DADS on histone acetyla-
tion in vivo in normal colonocytes isolated from rats. The
results showed that DADS was capable of inducing hyper-
acetylation of histones H4 and H3 in colonocytes after in
vivo administration [Druesne-Pecollo et al., 2007]. More-
over, their experiments revealed the ability of DADS to
modulate expression of a few genes in colonocytes in
vivo. They performed analysis of gene expression profiles
on the RNA extracted from rat colonocytes isolated 1 or
21 hr after the end of the intracaecal perfusion. That study
evidenced that as early as 1 hr after the end of the perfu-
sion, DADS affected the mRNA level of some proteins
(e.g., glutathione S-transferase p class, mitogen-activated
protein kinase 3, inhibitor of DNA binding 1). Twenty-
one hours after the end of intracaecal perfusion, the
expression of 49 various genes involved in several proc-
esses including cell cycle, DNA repair and cellular adhe-
sion factors seemed to be modified by DADS [Druesne-
Pecollo et al., 2007]. Those authors observed a correlation
between the modulation of gene expression and histone
hyperacetylation in response to DADS.
All studies presented in this chapter suggest that OSCs-
induced histone hyperacetylation could be one of the
mechanisms involved in their chemoprotective properties.
Mechanism of Organosulfur Compounds-InducedApoptosis
Most often two pathways leading to apoptosis are dis-
tinguished: intrinsic (mitochondrial) and extrinsic (death
receptor) [Kaufmann and Hengartner, 2001]. Both path-
ways of apoptosis comprise a family of intracellular pro-
teases, called caspases.
The final stages of apoptosis in mitochondrial pathway
are crucially dependent on caspase 3, which acts as the
executioner for cell death by cleaving multiple structural
and repair proteins, e.g., cleaving of poly(ADP-ribose)
polymerase (PARP) [Kaufmann and Hengartner, 2001;
Slee et al., 2001]. The main regulators of this pathways
Environmental and Molecular Mutagenesis. DOI 10.1002/em
256 Iciek et al.
are members of the Bcl-2 protein family, which consists
of antiapoptotic proteins, (e.g., Bcl-2, Bcl-xL), and pro-
apoptotic proteins, (e.g., Bax, Bad, Bak, Bik, Bid). Intra-
cellular ratio of antiapoptotic/pro-apoptotic members of
Bcl-2 family can serve as a marker of cell sensitivity to
apoptotic stimuli [Farrow and Brown, 1996].
The other pathway, called extrinsic or death receptor
pathway relies on the ligand-activated recruitment of adap-
tor proteins by death receptor. When membrane receptors,
like Fas or TNFR1, bind their selective ligands, they ex-
pose death domains responsible for binding of proteins
possessing similar domains to form signaling complexes.
These complexes serve to activate the initiator caspases
(e.g., caspase 8), which then initiate proteolytic cascade,
activating the executioner caspases (e.g., caspase 3, 6, 7).
Next, they activate nucleases, and planned degradation of
cell components progresses [Kaufmann and Hengartner,
2001].
Molecular mechanisms involved in the induction of
apoptosis and caspase activation by garlic-derived organo-
sulfur compounds are complex and only fragmentarily
known. Studies have shown that OSCs stimulate the mito-
chondrial apoptotic pathway. For instance, DADS induced
apoptosis in estrogen receptor-positive and -negative
human breast cancer cells, and it was correlated with up-
regulation of Bax and down-regulation of Bcl-xL [Naka-
gawa et al., 2001]. A change in intracellular Bcl2/Bax
ratio was also observed in DAS-, DADS-and garlic
extract-treated lung cancer cells [Hong et al., 2000]. Stud-
ies of Kwon et al. demonstrated that DADS induced apo-
ptosis of human leukemia HL-60 cells in a concentration-
and time-dependent manner. DADS activated caspase-3 as
evidenced by both the proteolytic cleavage of the proen-
zyme and increased protease activity. The activation of
caspase-3 led to the cleavage of poly(ADP-ribose)poly-
merase (PARP). Those authors also revealed that DADS
increased the production of intracellular hydrogen perox-
ide. In that study DADS-induced apoptosis was prevented
by preincubation with catalase and by the presence of
exogenous antioxidants, such as N-acetylcysteine [Kwon
et al., 2002]. This suggests that DADS-induced apoptosis
is mediated by reactive oxygen species (ROS), but the
detailed mechanisms remain to be elucidated.
Lu et al. in their studies conducted on human bladder
cancer cells T24 obtained the same results, namely, DADS
induced apoptosis through the activation of the mitochon-
drial pathway: Bcl-2 down-regulation, cytochrome c release
into the cytosol, activation of caspase-9 and caspase-3.
The increased caspase-3 activity in DADS-treated T24
cells was accompanied by PARP degradation and increase
in intracellular hydrogen peroxide production [Lu et al.,
2004]. An involvement of reactive oxygen species in apo-
ptosis induction by OSCs was additionally documented by
the studies of Filomeni et al. using a neuroblastoma cell
line SH-SY5Y. Those authors observed that the earliest
oxidative event after DADS administration was an
increase in ROS production, which reached the maximum
yield at 30 min after DADS treatment [Filomeni et al.,
2003]. Apart from a commonly known role of ROS in
lipid peroxidation and protein damage, reactive oxygen
species can also participate in redox-mediated signal
transduction. It was demonstrated that DADS induced cell
death via a redox-mediated process involving ROS pro-
duction, which resulted in a transient oxidative damage to
proteins and lipids, and to the activation of the JNK/c-Jun
transduction pathway. Wen et al. reported that DADS
induced a temporary increase in phosphorylated p38
MAPK and phosphorylated p42/44 MAPK in HepG2 cells
[Wen et al., 2004]. MAPKs (mitogen-activated protein
kinases) are serine-threonine protein kinases that are acti-
vated in response to a diverse range of stimuli including
growth factors, hormones, neurotransmitters as well as
cellular stress. P38 MAPK, c-Jun kinase (JNK) and p42/
44 MAPK are classified as subfamilies of MAPK. MAP
kinases are known to be modulated by ROS, in particular,
neuronal cell death is often mediated by the activation of
JNK, the c-Jun upstream MAP kinase [Herdegen and
Waetzig, 2001]. The basal activity of JNK is maintained
low through the formation of a heterocomplex with gluta-
thione-S-transferase (GST), which can bind JNK thereby
limiting the degree of c-Jun phosphorylation and inhibi-
ting its kinase activity [Adler et al., 1999]. Under oxida-
tive stress, GST/JNK complex dissociates leading to JNK
activation. Filomeni et. al. found that DADS treatment
induced a rapid dissociation of GST from JNK). These
authors suggested that ROS production played a crucial
role in DADS-induced apoptosis, especially in cells pos-
sessing a poor antioxidant defense, like tumor cells [Filo-
meni et al., 2003].
Xiao et al. investigated the mechanisms involved in ap-
optosis induced by DATS in human prostate cancer cells
PC-3 and DU145. The studies revealed that DATS was a
significantly more potent inducer of cell apoptosis than
DAS and DADS. DATS-induced apoptosis in PC-3 cells
was associated with phosphorylation of Bcl-2, reduced
Bcl-2:Bax interaction and activation of procaspase-9 and -
3. They found that DATS treatment resulted also in the
activation of extracellular-signal regulated kinase ERK1/2
and c-jun N-terminal kinase 1 (JNK1). Overexpression of
catalase, one of the main hydrogen peroxide scavengers,
inhibited DATS-induced JNK activation, and apoptotic
death. It suggests that these events are mediated through
oxidative stress, and hydrogen peroxide can act as a sec-
ond messenger [Xiao et al., 2004]. The authors postulated
that DATS treatment increased the intracellular level of
ROS, which can be detected by redox-sensitive molecules
including thioredoxin (Trx) and glutaredoxin (Grx). These
proteins bind to apoptosis signal-regulating kinase 1
(ASK 1) and suppress its activation. During oxidative
stress Trx and Grx dissociate from ASK 1 and this event
Environmental and Molecular Mutagenesis. DOI 10.1002/em
Anticancer Properties of Garlic 257
activates the ASK1-SEK1-JNK1 signal transduction path-
way [Song and Lee, 2003].
Therefore, implication of oxidative stress in the induc-
tion of apoptosis by garlic-derived organosulfur com-
pounds seems sufficiently documented. However, the
question arises how OSCs induce oxidative stress thereby
contributing to the activation of apoptosis-triggering sig-
nal transmission pathways.
According to Munday et al., di-, tri- and tetrasulfides
can generate hydrogen peroxide in the presence of GSH
and oxygen bound to hemoglobin [Munday et al., 2003].
Disulfide (e.g., DADS) can be readily reduced by GSH to
a thiol (reaction 1), which dissociates to thiolate anion
(reaction 2). The resulting thiolate anion undergoes one-
electron oxidation in the presence of transition metal ions,
such as Fe31, generating thiyl radical (reaction 3).
RSSR þ 2GSH ! 2RSH þ GSSG ð1Þ
RSH $ RS� þ Hþ ð2Þ
RS� þ Fe3þ ! RS� þ Fe2þ ð3Þ
Fe2þ þ O2 ! Fe3þ þ O��2 ð4Þ
O��2 þ HO�
2 ! H2O2 þ O2 ð5Þ
H2O2 þ Fe2þ ! �OH þ �OH þ Fe3þ ð6Þ
Reduced metal ions (Fe21) react with molecular oxygen
to produce superoxide radical anion (reaction 4), which
can react with hydroperoxide radical generating hydrogen
peroxide and molecular oxygen (reaction 5). The latter
can also participate in the Fenton reaction, generating
highly reactive hydroxyl radical (reaction 6).
Thiyl radical, formed in reaction 3, is reconverted to di-
sulfide either through dimerization (reaction 7) or via the
formation of disulfide radical anion (reaction 8). The lat-
ter can also react with molecular oxygen to produce
superoxide radical anion (reaction 9):
2RS� ! RSSR ð7Þ
RS� þ RS� ! RSSR�� ð8Þ
RSSR�� þ O2 ! RSSR þ O��2 ð9Þ
It can be concluded that disulfides are reduced by GSH to
thiols, which can generate ROS under biologically rele-
vant conditions in the presence of transition metal ions
via a sequence of reactions, which can be summarized as
follows:
2RSH þ O2 �!Fe3þ=Fe2þ
2RS� þ H2O2 ð10Þ
Because a carbon-sulfur bound of monosulfides (e.g.,
DAS) is not cleaved by mild reducing agents, like GSH,
thiol formation from this compound does not occur. This
explains why there are no literature reports about DAS-
induced increase in ROS production.
In contrast, trisulfides (DATS) can be readily cleaved
by GSH, yielding a mixture of perthiol, thiol and glutathi-
one disulfide (reaction 11) or alternatively perthiol and
mixed disulfide (reaction 12).
RSSSR þ 2GSH ! RSSH þ RSH þ GSSG ð11Þ
RSSSR þ GSH ! RSSH þ RSSG ð12Þ
Thiols dissociate and enter a sequence of reactions
described above. However, the resulting perthiols dissoci-
ate forming perthiyl anions (reaction 13), which can be
oxidized by traces of transition metal ions present in the
aqueous buffer to perthiyl radicals (reaction 14). The
reduced metal ions can reduce molecular oxygen to super-
oxide radical anion and other reactive oxygen species,
according to reactions 4–6.
RSSH $ RSS� þ Hþ ð13Þ
RSS� þ Fe3þ ! RSS� þ Fe2þ ð14Þ
The persulfide radicals formed in reaction 14 are thought
to be relatively stable species. They can dimerize to gen-
erate a polysulfide (reaction 15), which can be different
than the initial compound (here tetrasulfide). Alterna-
tively, the persulfide radicals may react with GSH to form
a polysulfide radical anion, which may reduce molecular
oxygen to yield ROS and regenerate a polysulfide (reac-
tions 16–17).
2RSS� ! RSSSSR ð15Þ
RSS� þ GSH ! RSSSG�� ð16Þ
RSSSG�� þ O2 ! O��2 þ RSSSG ð17Þ
It can be summarized that trisulfides upon reduction to
perthiols and thiols can generate ROS (H2O2, O2�2, �OH)
under physiological conditions by reacting with molecular
oxygen in the presence of transition metal ions (reaction
18).
2RSSH þ O2 �!Fe3þ=Fe2þ
2RSS� þ H2O2 ð18Þ
Compared to thiols (RSH), perthiols (RSSH) are more
acidic, hence, their larger fraction exists in the reactive
anionic form. RSSH are also strong reducing agents,
which react rapidly with oxidants, such as molecular oxy-
gen to form reactive oxygen species. The differences
between di-(e.g., DADS) and trisulfides (e.g., DATS) in
the capability of ROS generation and inability of mono-
sulfides (e.g., DAS) to form ROS can explain their dispar-
ate toxicities. Many authors noted that the toxicity of ana-
logical doses of different OSCs decreases in the following
order DATS > DADS > DAS [Munday et al., 2003].
Environmental and Molecular Mutagenesis. DOI 10.1002/em
258 Iciek et al.
Some studies have suggested that apoptosis induction
by OSCs can be connected with an increase in free intra-
cellular calcium (Ca21) level. Disruption of cellular
Ca21 homeostasis (e.g., altered extrusion or transport)
can lead to apoptosis. Studies of Sundaram et al. con-
ducted on colon tumor cell line (HCT-15) revealed that
DADS, but not SAC induced a dose-dependent increase
in intracellular Ca21 levels, which was observed as early
as 4 min after DADS treatment [Sundaram and Milner,
1996a]. Similar increase in Ca21 level in some cells was
also reported after treatment with other sulfides [Hua
et al., 1993]. Moreover, the treatment of HCT-15 cells
with DADS resulted in a dose-dependent decrease in the
activity of calcium-dependent ATPase. Sakamoto et al.
observed a marked and progressive increase in intracellu-
lar Ca21 level after treatment with DATS in human
A549 lung tumor cells. After refeeding of complete me-
dium without DATS, the intracellular Ca21 level in
A549 cells returned to near control levels [Sakamoto
et al., 1997].
Park et al. studied the role of Ca21 in DADS-induced
apoptosis of HCT-15 cells. Upon DADS exposure there
was a rapid and sustained increase in Ca21 level, which
was connected with an enhanced production of hydrogen
peroxide and caspase-3 activation. Moreover, the treat-
ment of HCT-15 cells with BAPTA, an intracellular Ca21
chelator, abolished DADS-induced Ca21 elevation and
hydrogen peroxide production, which further prevented
caspase-3 activation and DNA fragmentation. The authors
suggested that the increase in Ca21 level was a key medi-
ator in DADS-induced apoptosis in HCT-15. Because no
increase in calcium level was observed in HL-60 cells,
they concluded that the disruption of Ca21 homeostasis
was not a universal phenomenon, but rather was depend-
ent on the cell type [Park et al., 2002].
More recently Karmakar et al. have reported that both
DADS and DAS elevated Ca21 level in human malignant
neuroblastoma SH-SY5Y cells, which led to the activation
of caspase-3 and caspase-9 linked with the intrinsic path-
way of apoptosis. Moreover, they observed that elevated
Ca21 level caused calpain activation in SH-SY5Y cells
[Karmakar et al., 2007]. Calpain is a Ca21-dependent
noncaspase cysteine protease that can contribute to cell
death by inducing the mitochondrial apoptosis pathway
independently of caspases. Calpain can produce a potent
pro-apoptotic Bax fragment to trigger Bcl-2-independent
cytochrome c release from mitochondria thereby inducing
apoptosis [Gao and Dou, 2000].
A great majority of studies devoted to anticancer action
of garlic-derived organosulfur compounds was conducted
in vitro. Although results of these studies clearly indicate
that some garlic compounds show anticancer properties,
not always these effect can be observed in vivo. More-
over, often there is a problem with reaching a compara-
ble, effective concentration in in vivo conditions.
S-Thiolation Reactions
Pinto and coworkers have suggested that garlic-derived
organosulfur compounds can modify ��SH-containing
enzymes via thiol-disulfide exchange similar to that of
protein glutathionylation [Pinto et al., 2006]. The S-thiola-
tion reaction relies on formation of a mixed disulfide
between a protein thiol and a low molecular weight thiol,
e.g., glutathione or cysteine. This reversible post-transla-
tional modification of protein is believed to be an antioxi-
dant mechanism, since under oxidative conditions it
affords reversible protection to ��SH groups against their
irreversible oxidation to sulfonic acids, that leads to the
loss of biological activity of proteins. The formation of
mixed disulfides with proteins is also a redox method reg-
ulating protein activity and a mechanism of signal trans-
duction in the cell [Biswas et al., 2006]. Cysteine ��SH
groups in protein can exist as fully reduced or partially
and reversibly oxidized (e.g., sulfenic acid), which pro-
vides the means to precisely regulate biological activity of
these molecules. It is well known that reactive oxygen spe-
cies fulfill a regulatory role in the cell, while reversible S-
thiolation can be considered to be a regulatory redox
mechanism for cellular processes. Depending on the intrin-
sic nature of a protein or an enzyme, S-thiolation may ei-
ther activate or inactivate it [Klatt and Lamas, 2000].
Rabinkov investigated the reaction between diallyl thio-
sulfinate (allicin) and reduced glutatione. The product of
this reaction S-allylmercaptoglutathione (SAMG) reacted
with SH-containing enzymes, papain and alcohol dehydro-
genase yielding corresponding S-allylmercaptoproteins,
that caused inactivation of these enzymes. The activity
was restored by ditiothreitol [Rabinkov et al., 2000]. Alli-
cin can similarly react with reduced cysteine yielding
SAMC.
There are two routes of S-thiolation: either via thiol-di-
sulfide exchange or via the reaction of reversibly oxidized
sulfhydryl groups (e.g., sulfenic acid) with low-molecular-
weight thiols. It seems that the first route is more proba-
ble for garlic-derived biologically active compounds.
Disulfides, like DADS, can react with protein sulfhydryl
groups forming mixed disulfides (reaction 19).
allyl-SS-allyl þ protein-SH ! protein-S-S-allyl þ allyl-SH
ð19Þ
In the case of trisulfides, e.g., DATS, the reaction with
protein thiols can result in a mixed disulfide and a persul-
fide (reaction 20):
allyl-S-S-S-allyl þ protein-SH
! protein-S-S-allyl þ allyl-S-SH ð20Þ
Similar thiol-disulfide exchange reaction can occur
between protein sulfhydryl groups and S-cysteinyl com-
pounds from garlic, such as SAMC (reaction 21):
Environmental and Molecular Mutagenesis. DOI 10.1002/em
Anticancer Properties of Garlic 259
allyl-SS-Cys þ protein-SH ! protein-S-S-Cys þ allyl-SH
ð21Þ
Pinto et al. have suggested that such S-cysteinylation of
signaling proteins and transcription factors may be a pri-
mary target for development of chemopreventive or thera-
peutic agents that stimulate pro-apoptotic proteins or inac-
tivate oncogenic factors [Pinto et al., 2006].
Apart from the transformation of garlic polysulfides by
a direct reaction of thiol-disulfide exchange, S-thiolation
via the second mechanism also cannot be excluded. As al-
ready mentioned, garlic derived di-tri-and tetrasulfides can
generate reactive oxygen species under physiological con-
ditions. Then, ROS can initiate S-thiolation by reacting
directly with protein ��SH groups to form protein thiyl
radicals (protein-S�) or sulfenic acids (protein-SOH) (reac-
tions 22–23). These reactive forms of thiols can then react
with low-molecular-weight thiols (GSH, cysteine, allyl
mercaptan), leading also to the formation of mixed disul-
fides, i.e., to S-thiolation (reactions 24–25).
protein-SH þ R� ! protein-S� þ RH ð22Þ
protein-SH þ H2O2 ! protein-SOH þ H2O ð23Þ
protein-S� þ RSH ! protein-S-S-R þ H� ð24Þ
protein-SOH þ RSH ! protein-S-S-R þ H2O ð25Þ
There are literature reports of functional regulation of many
proteins by S-thiolation [Klatt and Lamas, 2000]. Important
examples of such proteins are: proteins involved in cellular
signaling, like protein phosphatases, protein kinases,
NFjB, c-Jun/AP-1, p53 and H-ras [Biswas et al., 2006].
Hence, the involvement of garlic-derived polysulfides
in S-thiolation is very probable [Munchberg et al., 2007]
though insufficiently confirmed experimentally.
Modifications With Sulfane Sulfur
Organosulfur compounds from garlic can also be
engaged in the modifications of protein sulfhydryl groups
by sulfane sulfur, which yields perthiols or trisulfides.
Sulfane sulfur is a highly reactive sulfur atom in the
reduced form. It has an oxidation state of 0 or 21 and is
covalently bound to another sulfur atom. Sulfur with such
features can easily leave the compound’s structure and
can be transferred to such acceptors as thiols or cyanide
[Iciek and Włodek, 2001]. Examples of the compound
containing sulfane sulfur are perthiols (RSSH), polysul-
fides (R-Sn-R, where n � 3), thiosulfate. Another group
of compounds with sulfane sulfur includes disulfides con-
taining a double bond and carbonyl or enol group in the
molecule, which enables them to tautomerize to sulfane-
sulfur-containing thiosulfoxides [Toohey, 1989]. Thus,
DADS from garlic can be a source of the labile sulfane
sulfur (Scheme 5). DATS and higher polysulfides can be
direct sulfane sulfur donors whereas DADS can acquire
this ability by tautomerization.
We have suggested that the antiproliferative DADS action
on human hepatoma cell line (HepG2) depends on the pres-
ence of the sulfane sulfur. We observed similar but less
potent antiproliferative effects of other systems, which can
yield sulfane sulfur compounds, on HepG2 cells [Iciek
et al., 2001]. Our other in vivo studies proved that DADS
was able to efficiently increase sulfane sulfur level and ac-
tivity of sulfotransferases implicated in its metabolism in the
liver of ascites tumor-bearing mice but it did not affect can-
cer cells [Iciek et al., 2007]. It indicates that this compound
can elicit an efficient and selective beneficial hepatoprotec-
tive effect, what can be helpful in chemotherapy. Moreover,
we found that DADS, being a sulfane sulfur donor,
increased the number of brain Gomori positive cytoplasmic
granulations, that can be a source and a store of sulfane sul-
fur in mammals [Iciek et al., 2005, Srebro et al., 2008].
Cooper and coworkers investigated enzyme-catalyzed
transformations of several allium-derived L-cysteine-S-con-
jugates. They observed that SAC, SAMC and S-propylmer-
capto-L-cysteine are substrates of cysteine S-conjugate
b-lyase [Cooper and Pinto, 2005]. Cysteine S-conjugate
b-lyases are PLP-containing enzymes that catalyze b-elimi-
nation reactions with cysteine S-conjugates. The end prod-
ucts of this reaction are pyruvate, ammonium and a sulfur-
containing fragment (reaction 26).
Cys-S-R þ H2O ! pyruvate þ NHþ4 þ RSH ð26Þ
Those authors have suggested that g-cystathionase is a
major enzyme responsible for the cysteine S-conjugate b-
lyase reactions in the rat liver cytosol. This cytosolic
enzyme whose biological role consists in cystathionine
cleavage to cysteine, catalyzes also cystine cleavage to thi-
ocysteine, which is an unstable compound and is easily
transformed into a stable thiocystine trisulfide. The prod-
ucts of both reactions contain sulfane sulfur. Cooper and
Pinto suggested that the b-lyase reaction catalyzed by g-
cystahionase on allium-derived SAMC could yield sulfane
sulfur containing perthiols (RSSH) (reaction 27).
allyl-S-S-Cys �!cystathionaseallyl-SSH þ NHþ
4 þ pyruvate
ð27Þ
Cystathionase catalyzes similar b-lyase reaction utilizing
other allium-derived cysteine S-conjugates, like S-propyl-
Environmental and Molecular Mutagenesis. DOI 10.1002/em
Scheme 5. Diallyl disulfide tautomerization into thiosulfoxide contain-
ing a sulfane sulfur atom.
260 Iciek et al.
cysteine and S-penta-1,3-dienylmercaptocysteine [Cooper
and Pinto, 2005]. Biological effects of these compounds
can be underlain by the addition of sulfane sulfur to cyste-
ine residues of redox-sensitive proteins (np. NFjB, P53)
catalyzed by rhodanese. The main physiological function of
rhodanese consists in transport of the reactive sulfane sulfur
from anionic donors (thiosulfate, perthiols, polysulfides) to
thiophilic acceptors (cyanide, thiols, sulfate (IV)). Covalent
modification of protein ��SH groups by sulfane sulfur
yielding trisulfides and perthiols plays a significant regula-
tory role. Organosulfur compounds derived from garlic,
being a source of sulfane sulfur, can interact with cysteine
thiol groups in catalytic, structural or regulatory proteins.
These modifications induce changes in the intracellular
redox potential and can influence cancer cell growth by
targeting sulfhydryl groups of redox-sensitive signaling
proteins, and in this way regulate cell proliferative and apo-
ptotic responses.
Other Effects of Organosulfur Compounds
Interestingly, Huard et al. have studied in vitro the
effect of DADS on gap-junctional intercellular communi-
cation (GJIC) [Huard et al., 2004]. Gap junctions are
transmembrane channels composed of proteins called con-
nexins. They permit neighboring cells to communicate
directly by sharing small cytoplasmic molecules, like ions
and second messengers [Evans and Martin, 2002]. Many
evidences suggest that an alteration in gap-junctional
intercellular communication is involved in tumor cell
development [Trosko and Ruch, 1998]. Some studies
revealed that DADS was able to improve GJIC in rat liver
epithelial cells. This stimulation was time-dependent, in
addition, incubation of the cells with DADS for 1 hr pre-
vented tumor promoter 3,5-di-tertio-butyl-4-hydroxyto-
luene-induced inhibition of GJIC. The effect of DADS on
rat liver epithelial cells was attributed to its ability to
enhance the amount of connexin 43, the main connexin
expressed in these cells. Moreover, DADS induced rapid
inhibition of protein glycosylation in that study [Huard
et al., 2004]. The ability of DADS to enhance GJIC is an
interesting property of this compound and possibly its
other derivatives, which may be important for anticarcino-
genic activities of OSCs.
Other studies have shown that OSCs present in garlic
can modulate the expression of two transporters involved
in the cell defense and in the development of multidrug
resistance. Multidrug resistance is a resistance of tumors
to anticancer drugs and it is one of the major problems of
anticancer chemotherapy. Although multiple mechanisms
mediate multidrug resistance, there are two main proteins
involved in establishing the multidrug resistance in cancer
cells: P-glycoprotein (P-gp) and multidrug resistance pro-
tein 2 (Mrp2). P-gp is an ATP-dependent transporter,
which mediates resistance to various classes of chemo-
therapeutic agents by actively extruding the drugs from
the cells and lowering their intracellular concentrations
[Gottesman and Pastan, 1993]. Mrp2 is an ATP-dependent
transporter for organic anions that contributes to the drug
resistance by transporting a wide range of glutathione,
glucuronate and sulfate conjugates out of cells. Studies of
Demeule et al. indicated that DADS and SAC modulated
the expression of both Mrp2 and G-pg in rat renal brush-
border membrane [Demeule et al., 2004]. On the other
hand, Nabekura et al. investigated the effect of various
dietary phytochemicals, among them DAS and DATS on
P-gp function using human multidrug-resistant carcinoma
KB-C2 cells. They found that DAS and DATS had no
effect on P-gp in these cells [Nabekura et al., 2005]. The
results obtained by Arora et al. showed that DAS
decreased the elevated levels of P-gp in resistant leukemic
cells K562 back to the normal levels [Arora et al., 2004].
Concluding Remarks
Garlic is a commonly known natural medicinal product,
used in the treatment of many diseases. Owning to diver-
sity of its action (e.g., antibacterial, antifungal, antivirus,
cardiovascular properties), Powolny and Singh in their
last review proposed to name the multidirectional effects
of garlic as ‘‘promiscuous’’ and compared them with
diversified pharmacological aspects of aspirin [Powolny
and Singh 2008].
Epidemiological studies have indicated that the diet rich
in garlic and other plants of the family Allium decreases
risk of some types of cancer. Numerous laboratory studies,
some of which were cited in this review, indicate anti-
cancer properties of garlic or garlic-derived sulfur com-
pounds. Garlic-derived organosulfur compounds produce
the anticancer effects by influencing drug metabolizing
enzymes inhibiting tumor initiation, eliciting antioxidant
activity, inducing apoptosis and cell cycle arrest, and by
modulating signal transduction.
As shown in this review, the mechanisms of anticancer
action of garlic-derived OSCs are multidirectional and it
is difficult to find a common target. It is still unknown
how these anticancer properties of garlic OSCs from in
vitro studies can be used in clinical practice. People most
often eat garlic, whereas a majority of laboratory studies
have examined individual organosulfur compounds. More-
over, as mentioned in this review, there is some evidence
of high toxicity of large doses of raw garlic. Thus,
attempts have been made to isolate individual biologically
active and relatively safe compounds and their preparation
for oral use.
Therefore, future studies should focus most of all on
four aspects of anticancer activity of garlic and its prepa-
rations. Firstly, details of metabolic transformations of
garlic and garlic-derived compounds in human body
should be clarified. Secondly, the question arises whether
Environmental and Molecular Mutagenesis. DOI 10.1002/em
Anticancer Properties of Garlic 261
the efficient concentration of OSCs used in in vitro cul-
tures can be achieved in humans. Thirdly, knowledge of
toxicology of individual OSCs is needed to develop a safe
therapy. Fourthly, the mechanism of selective induction
of apoptosis by OSCs in cancer cells without any effect
on normal cells, remains to be elucidated.
Such thorough analysis can help researchers choose the
maximally efficient and minimally toxic composition of
garlic preparations.
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