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Introduction
Health benefits of Allium vegetables including garlic have
been noted through out recorded history, dating back to 1400
BC[1]. The known medicinal benefits of garlic and other
Allium vegetables and their constituents include lowering
of serum cholesterol level, inhibition of platelet aggregation
and increased fibrinolysis [2,3], stimulation of immune
function through activation of macrophages and induction of
T-cell proliferation[4,5], reduction of blood glucose
level[6,7],
radioprotection[8], improvement of memory and learning
deficit[9,10], protection against microbial, viral and fungal
infections[11_13], and anticancer
effects[14,15]. Initial evidence for
the anticancer effect of Allium vegetables was provided by
population-based observational
studies[16-18]. For example, You et
al[16] documented a significant reduction in gastric
cancer risk with increasing intake of Allium vegetables in a population-based, case-control study. Likewise,
Steinmetz et al[17] observed an inverse correlation between fruit and
vegetable intake and colon cancer risk in the Iowa Women's
Health Study.
The sulfur chemistry of garlic is fairly well
understood[19]. The main sulfur compound in intact garlic is
g-glutamyl-S-alk(en)yl-L-cysteine, which is hydrolyzed and oxidized to yield
alliin[19]. Alliin accumulates naturally during storage of the
bulbs at cool temperature and is the odorless precursor of
the organosulfur compounds (OSC) believed to be
responsible for the anticancer effect of
garlic[20_24]. Processing of garlic bulbs (crushing, cutting or chewing) releases a
vacuolar enzyme alliinase that acts on alliin to give rise to
extremely unstable and odoriferous compounds, including
allicin. Allicin and other thiosulfinates decompose to
oil-soluble OSC, including diallyl sulfide (DAS), diallyl
disulfide (DADS), diallyl trisulfide (DATS), dithiins and ajoene
(4,5,9-trithiadodeca-1,6,11-triene-9-oxide)[19]
.
Preclinical animal studies have indicated that OSC
analogues are highly effective in affording protection against
cancer induced by a variety of chemical
carcinogens[20_24]. For instance,
Belman[20] demonstrated that topical
application of garlic and onion oil inhibited the incidence of tumor
promoted by phorbol_myristate_acetate. Cancer
chemopre-vention by garlic constituents has been observed against
benzo[a]pyrene (BP)-induced forestomach and pulmonary
cancer in mice[21], N-nitrosomethylbenzylamine-induced
esophageal cancer in rats[22], azoxymethane-induced colon
carcinogenesis in rats[23], and
2-amino-1-methyl-6-phenyli-midazo[4,5-b]pyridine-induced mammary tumorigenesis in
rats[24]. Elucidation of the mechanisms by which OSC may
offer protection against cancer has been a passionate
subject of research for the past 20 years. This article summarizes
current knowledge on the molecular targets of cancer
chemoprevention by garlic constituents.
Modulation of carcinogen activation
Carcinogenic activity of many environmental pollutants
(which are usually lipophilic substances) is often dependent
on their activation by cytochrome P450-dependent
monooxy-genases. Garlic constituent DAS and its metabolites diallyl
sulfoxide and diallyl sulfone competitively inhibited the
activity of cytochrome P-450 2E1 in a time-dependent and
NADPH-dependent manner using pseudo-first-order
kinetics[25]. Induction of cytochrome P-450 2B1 by treatment with
DAS in rat liver microsomes has also been
reported[25]. In rats treated with DAS after a 48-h fasting, the
starvation-induced hepatic microsomal P-450 2E1 level
decreased[26]. Moreover, DAS administration protected against
hepatotoxicity caused by exposure to P-450 2E1 substrates, including
N-nitrosodimethylamine (NDMA)[26]. The rat nasal cavity is
one of the known target organs for carcinogenesis by
NDMA, N-nitrosodiethylamine (NDEA) and tobacco
carcinogen 4-(methylnitrosamino)1-(3-pyridyl)-1-butanone
(NNK)[27]. A single po administration of DAS to male rats caused a
significant decrease in the oxidative metabolism of NDEA
and NNK in nasal mucosa[27]. Several naturally occurring
OSC, including DAS and DADS, inhibited the formation of
carcinogenic heterocyclic amines in boiled pork juice
(2-amino-3-methyl-imidazo[4,5-f]quinoline,
2-amino-3,4-dimethylimidazo[4,5-f]quinoxaline and
2-amino-3,4-dimethyli-midazo[4,5-f]quinoline)[28]
. In contrast, DAS, DADS and DATS have been shown to be inducers of rat liver
cytochrome P-450 1A1, 2B1 and 3A1[29]. Collectively,
modulation of carcinogen activation may be one of the mechanisms
by which garlic constituents may offer protection against
chemically induced cancers.
Induction of Phase 2 enzymes and modulation of anti-oxidative enzymes
Experimental evidence exists to suggest that garlic
constituents may function as a double-edge sword in the
prevention of chemically induced cancers by inhibiting
carcinogen activation and enhancing detoxification of activated
carcinogenic intermediates through the induction of Phase 2
enzymes, including glutathione transferases (GST) and
quinone reductase[30_33]. Wattenberg and colleagues showed
that prevention of BP-induced forestomach and lung cancer
in mice by garlic OSC was correlated with elevation of
hepatic and target organ total GST
activity[30]. Studies from our laboratory have shown that DAS, DADS and DATS
administration to A/J mice results in induced expression of
Alpha (mGSTA3-3, mGSTA1-2, mGSTA4-4), Mu (mGSTM1-1) and Pi class GST (mGSTP1-1) in the liver, lung and
forestomach[31_33]. However, OSC-mediated prevention of
BP-induced forestomach tumorigenesis, but not lung neoplasia,
in A/J mice is most closely correlated with the induction of
mGSTP1-1[31,32]. The DAS and DADS were found to be
potent inducers of quinone reductase activity and protein level
in the forestomach and/or lung of A/J
mice[34]. It is interesting to note that DATS administration only moderately
increased the activity of quinone reductase in the
forestomach or lung (about 1.5-fold increase compared with control
mice), despite a marked increase in its protein level, at least
in the forestomach[34]. Subsequently, Kong and colleagues
showed a positive correlation between OSC-mediated
induction of Phase 2 enzymes, activation of anti-oxidant response
element and accumulation of transcription factor nuclear
factor E2-related factor 2 in HepG2 hepatoma
cells[35]. Studies using Clone 9 liver cells documented an essential role for
GSTP enhancer I element (GPE I), but not GPE II, in
DADS-mediated and DATS-mediated induction of Pi class
GST[36]. Garlic-derived OSC have been shown to possess
non-enzymatic anti-oxidant activity[37]. The level of reduced
glutathione was increased in the liver, lung and/or forestomach
by DAS or DATS administration, but not by the non-allylic
OSC analogue dipropyl sulfide[38]. These OSC exhibited a
differential effect on the activities of glutathione redox cycle
enzymes in the liver, lung and forestomach of A/J
mice[38]. For instance, a noticeable increase in the activity of
glutathione peroxidase relative to control mice was observed
only in the lung of DATS-exposed
mice[38]. In contrast, Chen et
al[39] failed to observe a change in glutathione peroxidase
or superoxide dismutase activity in the liver, kidney, lung or
brain of rats treated with 50 or 200 mg/kg DAS for 8 or 29 d,
although hepatic catalase activity was significantly reduced.
DAS and DADS were shown to inhibit N-acetyltransferase
activity in a dose-dependent manner in a human colon
cancer cell line[40]. Thus, it is reasonable to conclude that the
induction of Phase 2 enzymes, especially GST, represents
another potential mechanism to explain OSC-mediated
prevention of chemically induced cancers. However, the
relationship between the chemopreventive effects of OSC and
their effects on anti-oxidant enzymes is somewhat
inconclu-sive.
Inhibition of post-translational modification of
oncogenic Ras
Studies from our laboratory have revealed that oral
administration of DADS (8.25, 16.5 and 33 µmol, 3 times per
week beginning the day of tumor cell injection), but not its
saturated analogue dipropyl disulfide, suppressed growth
of H-ras oncogene transformed tumor xenografts in nude
mice without causing weight loss or any other side
effects[41,42]. The appearance of measurable tumors was also delayed in
DADS-treated mice relative to
controls[41,42]. The DADS-mediated suppression of H-ras oncogene transformed
tumor growth correlated with a decrease in hepatic and
tumoral HMG-Co A reductase activity, leading to inhibition
of membrane association of p21[41,42]. In contrast, DADS
administration did not have any appreciable effect on
farnesyltrans-ferase activity in the
tumor[41,42]. These studies were the first published reports to document activity of
DADS against H-ras oncogene transformed tumors.
Inhibition of cell cycle progression
Cell cycle consists of a series of events involving growth
stimulus, replication and division of a eukaryotic
cell[43,44]. Cellular stresses may activate signal transduction pathways,
referred to as checkpoints, which lead to cell cycle
arrest[43,44]. The cell cycle checkpoints ensure completion of phase-
specific events and protect against genomic instability or, in
cases where the damage is too severe, switch the cell fate to
programmed cell death[43,44]. Many anticancer treatments
initially cause perturbations in cell cycle progression and
the interrupted phase depends on the genetic background
of the cell as well as the mode of action of a given treatment.
Studies have shown that garlic-derived OSC can suppress
growth of cancer cells of different anatomical locations in
association with cell cycle arrest, mainly in the G2/M phase
of the cell cycle. Milner and colleagues were the first to
show that DADS treatment caused dose-dependent and
time-dependent accumulation of human colon cancer cells in the
G2/M phase of the cell cycle[45,46]. The DADS-mediated
G2/M phase cell cycle arrest in human colon cancer cells was
accompanied by a decrease in the kinase activity of the
Cdk1/cyclin B1 complex, reduction in complex formation between
Cdk1 and cyclin B1, and a decrease in Cdc25C protein
level[46]. Some of these changes are not specific to colon cancer cells
or DADS because similar effects have been reported in other
cellular systems with other OSC[47_53]. For instance, DADS
(20 µmol/L, 12 h) caused inactivating phosphorylation of
Cdk1 in HL-60 cells[47] or decreased Cdk1 level in PC-3
human prostate cancer cells in a dose-dependent
manner[48].
We have tried to more thoroughly investigate the
mechanism of DATS-induced G2/M phase cell cycle arrest using
PC-3 and DU145 human prostate cancer cells as a
model[50_53]. DATS was much more effective than either DADS or DAS in
causing G2/M phase cell cycle
arrest[50]. These results further support the notion that even a subtle change in OSC
structure (the oligosulfide chain length) could have a
significant impact on its biological activity. Interestingly, a
normal prostate epithelial cell line PrEC was resistant to
growth inhibition and cell cycle arrest by
DATS[50]. The DATS-induced G2/M phase cell cycle arrest in PC-3 cells
was associated with increased Tyr15 phosphorylation of
Cdk1, inhibition of Cdk1/cyclin B1 activity, increased
inhibitory phosphorylation of Cdc25C at Ser216, and
downregula-tion of total Cdc25C protein
level[50]. The DATS-mediated hyperphosphorylation and decline in protein level of Cdc25C
were abrogated in the presence of anti-oxidants, suggesting
a redox-sensitive mechanism for these
effects[50]. We showed further that the Ser216 phosphorylation of Cdc25C was
mediated by Chk1, although its knockdown by Chk1-
specific siRNA was unable to rescue the G2/M phase block
caused by DATS[51]. In addition, the DATS-treated PC-3
cells exhibited features characteristic of mitotic arrest,
including changes in the tubulin network, chromatin
condensation and increased Ser10 phosphorylation of histone
H3[51]. Further examination of the DATS-treated PC-3 cells
revealed arrest in the prometaphase state that was partially
dependent on Chk1 activation and accompanied by
accumulation of anaphase promoting complex/cyclosome
(APC/C) substrates (cyclin A and cyclin B1), as well as
hyperphos-phorylation of securin and APC/C components (Cdc20 and
Cdh1)[52]. These results indicated that Chk1, which is an
intermediary of DNA damage
checkpoints[54], may regulate APC/C activity. Mitotic arrest has also been
documented for DADS and S-allyl mercaptocysteine
(SAMC)[55]. A schematic summary to explain the mechanism of DATS-induced
G2/M phase cell cycle arrest in human prostate cancer cells
is shown in Figure 1.
Recent studies from our laboratory have revealed that
DATS-mediated cell cycle arrest, at least in human prostate
cancer cells, is linked to c-Jun N-terminal kinase (JNK)-
dependent generation of reactive oxygen species
(ROS)[53]. The DATS-mediated ROS generation appears to be caused
by degradation of the iron-storage protein ferritin, which
leads to liberation of labile (chelatable)
iron[53]. The DATS-mediated degradation of ferritin, an increase in the labile iron
pool, ROS generation and the G2/M phase cell cycle arrest
are significantly attenuated by genetic suppression of
JNK[53].
Several studies show that OSC affect the microtubule
network in cancer cells that might initiate mitotic block or
apoptosis. For example, treatment of SW480 human colon
cancer cells or NIH3T3 mouse fibroblasts with 150 µmol/L
water-soluble SAMC caused rapid microtubule
depolymerization and cytoskeleton disruption in interphase
cells[56]. DATS, but not DADS or DAS, has been shown to induce
mitotic arrest in HCT-15 and DLD-1 human colon cancer cells
in association with disruption of the microtubule network in
interphase cells and inhibition of spindle formation in
mitotic cells[57]. This study further revealed DATS-mediated
oxidative modification of tubulin β at residues Cys12 and
Cys354[57]. Another oil-soluble garlic compound, Z-ajoene,
caused G2/M phase cell cycle arrest and disruption of the
microtubule network in normal marsupial kidney cells and
inhibited tubulin polymerization in
vitro[58].
A few reports have also shown that garlic-derived OSC
arrest cancer cells in phases other than G2/M. The
DADS-mediated suppression of human nosopharyngeal carcinoma
cell growth correlated with S phase
arrest[59]. Allitridi, synthetic DATS, was shown to arrest human gastric cancer
BGC823 cells in the G1 phase and was accompanied by a
decrease in cyclin D1 level and an increase in p27 protein
level[60]. Nevertheless, inhibition of cell cycle progression
appears to be a common cellular response to many
structurally diverse OSC.
Histone modification
OSC may affect cancer cell proliferation through
modification of histone acetylation and, thus, regulation of gene
expression. It has been reported that treatment of DS19
mouse erythroleukemia and K562 human leukemia cells with
DADS increases acetylation of histones H4 and
H3[61]. DADS and its metabolite, allyl mercaptan, inhibited histone
deacetylases in rat hepatoma and human breast cancer cells
and it has been suggested that histone acetylation may
medi-ate the differentiation process of erythroleukemia
cells[61]. Growth inhibitory effects of allicin, SAMC and S-allyl
cysteine (SAC) on DS19 cells and SAMC on Caco-2 human
colon and T47D human breast cancer cells are correlated
with increased histone acetylation[62]. The DADS-induced
accumulation of Caco-2 and HT-29 colon tumor cells in the
G2/M phase of the cell cycle is correlated with inhibition of
histone deacetylase, hyperacetylation of H3 and H4 histones,
and upregulation of p21 mRNA and protein
level[63,64].
Increase in p21 protein level with treatment of PC-3 cells with
DATS has also been documented, but antisense silencing of
p21 expression did not have any appreciable effect on
DATS-induced G2/M cell cycle arrest[50]. Whether or not p21
induction contributes to DADS-mediated G2/M phase cell
cycle arrest remains to be determined.
Induction of programmed cell death (apoptosis)
Apoptosis (also known as programmed cell death) is a
tightly controlled and evolutionarily conserved process of
cellular suicide critical to normal embryonic development and
maintenance of tissue homeostasis. Dysregulation of
programmed cell death underlies numerous pathological
conditions including cancer and, therefore, apoptosis is a valid
target in cancer therapy and
prevention[65,66]. Garlic-derived OSC have been shown to modulate a number of key
elements in cellular signal transduction pathways linked to the
apoptotic process. The majority of garlic-derived compounds
activate the so called intrinsic or mitochondria-mediated
pathway in the execution of apoptosis, which involves loss of
mitochondrial membrane potential and release of apoptogenic
molecules from the mitochondria to the
cytosol[67,68]. Activation of the intrinsic apoptotic pathway is regulated by the
Bcl-2 family of anti-apoptotic (eg Bcl-2 and Bcl-xL) and
pro-apoptotic (eg Bax and Bak)
proteins[69]. Garlic-derived OSC are believed to trigger apoptosis by modulating the levels of
Bcl-2 proteins. For example, DAS or DADS treatment
increased the ratio of Bax/Bcl-2 in SH-SY5Y neuroblastoma
cells, as well as in H460 and H1299 lung cancer cells
compared with untreated controls[70,71]. A time-dependent
upregulation of Bax protein level and concomitant
down-regulation of Bcl-xL protein level was observed in
DADS-treated MDA-MB-231 breast cancer cell
line[72]. The Z-ajoene-induced apopto-sis in HL-60 cells was associated with
caspase-mediated cleavage of Bcl-2[73]. Although Bcl-2
usually acts upstream of the caspase cascade its removal by
caspases may amplify the apoptotic signal. Cleavage of
Bcl-2 in Z-ajoene-treated cells was inhibited by anti-oxidants,
suggesting involvement of ROS in the activation of apoptosis
by this agent[73]. Indeed, a dose-dependent and
time-dependent increase in the production of peroxide was observed in
Z-ajoene-treated HL-60 cells[74]. We have shown that DATS
is a more potent inducer of apoptosis in PC-3 and DU145
prostate cancer cells than DAS or
DADS[75]. The DATS-induced apoptosis in prostate cancer cells correlates with a
decrease in Bcl-2 level as well as with hyperphosphorylation
of this protein, which reduces Bcl-2:Bax interaction and
activates the mitochondrial pathway of
apoptosis[75]. The DATS-mediated hyperphos-phorylation of Bcl-2 in PC-3 and
DU145 cells is caused by activation of JNK and, to a lesser
extent, extracellular signal-regulated kinase 1/2
(ERK1/2)[75]. Overexpression of Bcl-2 in PC-3 cells conferred statistically
significant protection against DATS-induced
apoptosis[75]. On the other hand, ectopic expression of Bcl-2 failed to
protect against DATS-mediated cell death in LNCaP human
prostate cancer cells[76], which unlike PC-3 are androgen
responsive and express wild type p53. Whether or not the
differential effect of Bcl-2 overexpression on DATS-induced
apopto-sis in PC-3 versus LNCaP is related to differences in their
androgen responsiveness or p53 status remains to be
investigated.
The DATS-induced apoptosis in LNCaP cells correlated
with a modest increase in protein levels of pro-apoptotic
Bcl-2 family members Bax and Bak[76]. The immortalized mouse
embryonic fibroblasts (MEF) derived from Bax and Bak double
knockout mice were found to be significantly more resistant
to DATS-induced apoptosis compared with the MEF derived
from wild-type mice[76]. Consistent with these results,
combined knockdown of Bax and Bak conferred statistically
significant protection against DATS-induced cell death in
LNCaP cells[76]. Furthermore, we showed that
DATS-mediated inhibition of PC-3 xenograft growth in nude mice
correlated not only with increased apoptosis but also with
induction of Bax and Bak proteins in the tumor
tissue[77]. However, it is important to point out that Bax and Bak cannot be
exclusively responsible for the cell death caused by DATS
because combined knockdown of these proteins conferred
only partial protection against DATS-induced
apoptosis[76]. It is intriguing that DATS treatment causes only a modest
increase in protein levels of Bax and Bak, yet knockdown of
these proteins confers statistically significant protection
against DATS-induced apoptosis[76]. Although the precise
mechanism by which Bax and Bak regulate DATS-induced
cell death remains elusive, it is possible that DATS treatment
causes conformation change and oligomerization of Bax/Bak
leading to their translocation to the mitochondria. This
possibility is likely based on the following considerations: (1)
Bax activation by certain apoptotic stimuli is dependent on
ROS generation, which is observed in DATS-treated
prostate cancer cells[76]; and (2) microtubule damaging agents
have been shown to cause Bax activation, and DATS
treatment is known to disrupt the tubulin
network[52]. However, further studies are needed to systematically explore this
possibility.
We have shown previously that the DATS-induced apoptosis in human prostate cancer cells was, at least in
part, regulated by the Akt-Bad
pathway[78]. One of the pro-survival functions of Akt (also known as protein kinase B) is
to phosphorylate Bad, which causes cytoplasmic
sequestration of Bad and consequently protection against
interaction with anti-apoptotic Bcl-2 family members. DATS
treatment markedly reduced Akt activity in PC-3 and DU145 cells
and consequently lowered the phosphorylation of Bad at
Ser155 and Ser136, which diminished complex formation
between Bad and cytosolic 14-3-3β[78]. Overexpression of
constitutively active Akt in PC-3 cells conferred significant
protection against DATS-induced
apoptosis[78]. The mechanism of DATS-induced apoptosis in human prostate cancer
cells is summarized in Figure 2.
Experimental evidence exists to support a critical role of
ROS as an intermediary of OSC-induced apoptosis. For
instance, DADS-induced apoptosis in HL-60 cells is
correlated with ROS generation[79]. The DADS-induced ROS
formation in SH-SY5Y neuroblastoma cells is evident as early
as 15 min after treatment and is accompanied by oxidation of
cellular lipids and proteins[80]. ROS generation in
DADS-treated cells was associated with activation of
JNK[80]. Over-expression of Cu,Zn-superoxide dismutase or pretreatment
with spin trapping molecule
5,5'-dimethyl-1-pyrroline N-
oxide offered protection against DADS-induced ROS generation, oxidative damage of cellular macromolecules and
apoptosis in SH-SY5Y cells[80].
A few studies have suggested that apoptosis induction
by OSC might result from an increase in free intracellular
calcium[70,81_84]. Park et
al[83] reported a biphasic response for
DADS-mediated elevation of calcium level with a rapid peak
at 3 min and slow and sustained elevation lasting up to 3 h
after the initiation of DADS treatment. The DADS-mediated
increase in intracellular calcium level was followed by an
increase in hydrogen peroxide level and caspase 3
activation[83]. Recently, it has been shown that both DAS and
DADS cause an increase in calcium level in SH-SY5Y cells,
which leads to activation of
calpain[70]. Calpain is a non-caspase cysteine protease that can contribute to cell death
by inducing mitochondria-mediated apoptosis independently
of caspases.
Some of the studies cited above have compared apoptotic
responses to OSC in cancer cells versus normal cells.
Strikingly, malignant cells appear to be more sensitive to
OSC-mediated apoptosis than normal non-transformed cells.
For example, viability of primary neurons was minimally
affected by treatment with 50 or 100 µmol/L DAS or DADS,
whereas the neuroblastoma of SH-SY5Y cells treated with
these concentrations of DAS or DADS exhibited a marked
reduction in cell viability[70]. Similarly, the viability of a
normal prostate epithelial line PrEC was not affected by DATS
treatment even at concentrations that are highly cytotoxic to
prostate cancer cells[50,76]. Finally, Z-ajoene has been shown
to cause apoptosis in human leukemia cells, but not in
peripheral mononuclear blood cells of healthy
donors[74]. The mechanism behind the differential sensitivity of cancer cells
and normal cells to apoptosis induction by OSC remains to
be elucidated.
Inhibition of angiogenesis and metastasis by
garlic constituents
Recent studies using cellular and animal models indicate
that garlic extract and its components are able to affect
tumor angiogenesis and metastasis. The formation of new
blood vessels is necessary for the growth of solid tumors
because evidence exists to suggest that tumor growth
beyond 1 mm in diameter is restricted by
angiogenesis[85]. A study by Matsuura
et al[86] showed that aged garlic extract
(AGE) suppressed proliferation of transformed human and
rat endothelial cell lines and reduced the invasiveness of the
endothelial cells by about 20%_30% as assessed by the
Matrigel chemoinvasion assay. Additional tests indicated
that AGE increased the adhesion of the endothelial cells to
collagen and fibronectin in a dose-dependent manner; thus,
reducing their motility[86]. Finally, AGE reduced
capillary-like tube formation by the endothelial cells in a
three-dimensional collagen matrix
assay[86]. We have examined the
effects of DAS, DADS and DATS on human umbilical vein
endothelial cell (HUVEC) viability and have shown that
DATS is the most potent of the three analogs in reducing the
viability of HUVEC[87]. The DATS-mediated suppression of
HUVEC proliferation correlated with caspase 3 and PARP
cleavage and apoptotic cell death[87]. The DATS treatment
was able to significantly disrupt the capillary-like tube
formation and migration by HUVEC that was accompanied by
suppression of vascular endothelial growth factor (VEGF)
secretion, downregulation of VEGF-Receptor 2 expression,
inactivation of Akt and activation of ERK
1/2[87]. In a follow-up study, we found that DATS administration to PC-3
prostate cancer-bearing male nude mice failed to inhibit the
formation of new blood vessels in the tumor as judged by
immunohistochemical staining for CD31, an endothelial cell
marker[77]. Alliin was shown to significantly reduce VEGF
and fibroblast growth factor 2- (FGF-2) induced tube
formation and angiogenesis in HUVEC and ex
vivo in CAM assay[88]. A recent study by Thejass
et al[89] showed that DADS and
DAS not only inhibited endothelial cell proliferation and
migration, but also reduced matrix metallopro-teinases 2 and
9. In addition, DAS administered to C57BL/6 mice injected
with B16F-10 melanoma cells increased circulating levels of
anti-angiogenic factors, tissue inhibitor of metalloproteinase
and interleukin-2 levels compared with the untreated
animals[90]. Attenuation of cell migration and the induction
of cell death by AGE was also documented in rat sarcoma
cells[91]. Taylor et
al[92] showed that ip injection of ajoene
(5_25 µg/g body weight) significantly inhibited pulmonary
metastasis in C57BL/6 mice injected with B16/BL6 melanoma
cells. Similarly, SAMC administration (300 mg/kg) to CB-17
SCID/SCID mice orthotopically implanted with PC-3 cells
reduced the number of lung metastasis per lung by 85.5%
and completely abolished adrenal gland metastasis, but had
no effect on local metastasis[93]. Based on the reviewed
studies it can be concluded that components of garlic extract (in
combination or alone) present a great potential as anti-angiogenic and antimetastatic agents.
Concluding remarks
Research over the past 20 years has revealed that
garlic-derived OSC can not only inhibit chemically induced
cancers but can also suppress growth of cancer cells in culture
and in vivo. The garlic compounds appear to target multiple
pathways, including the cell cycle machinery, the intrinsic
pathway for apoptotic cell death and angiogenic pathway,
which may all contribute to their anticancer activities.
Future research should focus on clinical assessment of these
compounds for prevention/treatment of cancers in humans.
A critical question relevant to the clinical development of
garlic OSC relates to their plasma or tissue concentration. It
remains to be determined whether the micromolar
concentrations of OSC needed to inhibit cancer cell growth in culture
are achievable in humans. It is important to point out that
the peak plasma concentration of DATS in rats following
treatment with 10 mg of the compound was shown to be
about 31 µmol/L[94]. Although the pharmacokinetic
parameters for DATS in humans have not yet been measured, oral
administration of 200 mg of synthetic DATS (also known as
allitridum) in combination with 100 µg selenium every other
day for 1 month to humans did not cause any harmful side
effects[95]. It is, therefore, possible that the plasma
concentrations of DATS required for cancer cell growth inhibition
may be achievable in humans.
Acknowledgements
The authors thank the past (X HU, H XIA, SK SRIVASTAVA, A PAL, J ANTOSIEWICZ, K LEW and Y
KIM) and present (D XIAO, H XIAO, E HAHM, S
STAN, Y ZENG, SW MARYNOWSKI, J A ARLOTTI and R WARIN)
members of the Singh laboratory for helpful discussion and
their contributions to the projects on cancer prevention by
garlic compounds.
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