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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