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Sulphur-containing non enzymatic antioxidants: therapeutic tools against cancer
Jose M. Mates, Juan A. Segura, Francisco J. Alonso and Javier Marquez
Department of Biologia Molecular y Bioquimica, Facultad de Ciencias, Campus de Teatinos, Universidad de Malaga, 29071
Malaga, Spain
1. Abstract
2. Introduction
3. Antioxidants and cancer
4. Thiol-containing compounds as antioxidant agents
5. Transsulfuration pathway
6. Cysteine
6.1. N-acetylcysteine
7. Homocysteine
8. Glutathione
8.1. Glutathione in the glutamate/glutamine network
8.2. Glutathione and drug resistance of cancer cells
9. Metionine
9.1. S-adenosylmethionine
10. Taurine
11. Lipoic acid
12. Summary and perspective
13. Acknowledgements
14. References
The prevention of oxidation is an essential
process in all cells, as decreased antioxidant protection may
lead to cytotoxicity, mutagenicity and carcinogenicity. The
mechanisms by which oxidative stress contributes to
carcinogenesis include modulation of gene expression and
induction of genetic modifications. Cellular methylation
and antioxidant metabolism are linked by the
transsulfuration pathway, which converts the methionine
cycle intermediate, homocysteine, to cysteine, the limiting
reagent in glutathione synthesis. Taurine can protect cells
from oxidant-induced injury scavenging strong oxidant and
cytotoxic agents, and lipoic acid can regenerate glutathione.
N-acetylcysteine has anticancer properties such as
counteractions against mutagens and prevention of tumor
progression. The oxidizing agents react with the thiol group
of these non enzymatic antioxidants determining cellular
redox potential, and modulating several biological events,
since different redox-sensitive molecules are involved in
many cell responses such as proliferation, growth arrest,
and death. The high metabolic activity characteristic of
cancer cells often upregulates oxidative stress protection
mechanisms. In fact, glutathione depletion is an early
hallmark observed in apoptosis and it has been
demonstrated as a common feature of cancer.
Under normal conditions, antioxidant systems of
the cell minimize the perturbations caused by reactive
oxygen species (ROS). When ROS generation is increased
to an extent that overcomes the cellular antioxidants, the
result is oxidative stress, which can be defined as the
imbalance between cellular oxidant species production and
antioxidant capability (1). Antioxidants are substances that
delay or prevent the oxidation of cellular oxidizable
substrates. The various antioxidants exert their effect by
scavenging ROS, or by activating of a battery of
detoxifying/defensive proteins (2). ROS thus play different
positive roles in vivo, being involved in energy production,
phagocytosis, cell growth and intercellular signalling
regulation. ROS may be also highly damaging, as they can
attack biological macromolecules, namely, lipids, proteins
and DNA, induce oxidation and cause membrane damage,
enzyme inactivation and DNA damage (3). However, when
the level of ROS exceeds the antioxidant capacity of the
cell, the intracellular redox homeostasis is altered and
oxidative stress ensues. Oxidative stress is considered to
play a pivotal role in the pathogenesis of aging and several
degenerative diseases such as cancer. In order to cope with
an excess of free radicals produced upon oxidative stress,
humans have developed sophisticated mechanisms to
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Thiol-non enzymatic antioxidants and cancer.
maintain redox homeostasis (2). These protective
mechanisms scavenge or detoxify ROS, block their
production, or sequester transition metals that are the
source of free radicals, and include enzymatic and
nonenzymatic antioxidant defenses produced in the body,
namely, endogenous, and others supplied with the diet,
namely, exogenous (4).
Several antioxidant enzymes exist that convert
ROS into less noxious compounds, for example, superoxide
dismutase (SOD), catalase,
peroxiredoxin and glutathione
Collectively, these enzymes provide a first line of defense
against superoxide anion (O2
(H2O2) (2). They are of enormous importance in limiting
ROS-mediated damages to biological macromolecules, but
they are not able to be 100% effective because certain
compounds generated by the interaction of ROS with
macromolecules are highly reactive (4). It is then
mandatory to detoxify these secondary products in to
prevent further intracellular damage, degradation of cell
components and eventual cell death (3). This second line of
defense against ROS is provided by enzymes such as
glutathione reductase (GR), glutathione S-transferase
(GST), aldo-keto reductase and aldehyde dehydrogenase.
The detoxified metabolites produced by these enzymes are
eliminated from the cell by efflux pumps such as the
glutathione S-conjugate transporter (4).
The third line of protection is carried out by no
enzymatic antioxidants. Many biologically important
compounds have been reported to have non enzymatic
antioxidant functions as vitamin C (ascorbic acid), vitamin
E (alpha-tocopherol), vitamin A (retinol), beta-carotene,
polyamines, melatonin (5-methoxy-N-acetyltryptamine),
nicotinamide adenine dinucleotide phosphate (NADPH),
urate, coenzyme Q-10 (ubiquinol), polyphenols, flavonoids,
phytoestrogens, terpenes, lipoic acid, glutathione (GSH),
cysteine, homocysteine, taurine, methionine, adenosine, S-
adenosyl-L-methionine (SAM), nitroxides and selenium
(3,5). Among the huge amount of antioxidant explored in
recent years, there are some of them showing important
effects on cancer (2). In this review we will focus on the
antioxidant function of the tripeptide glutathione, the
coenzyme alpha-lipoic acid (ALA), and the amino acids
cysteine, homocysteine, methionine, and taurine because
all have very low toxicological profiles, and they can
promote detoxification of
xenobiotics (6), promising an therapeutical action on cancer
through a redox-based strategy.
Antioxidants are considered as the most
promising chemopreventive agents against various human
cancers. However, some antioxidants play paradoxical
roles, acting as "double-edged sword." A primary property
of effective and acceptable chemopreventive agents should
be freedom from toxic effects in population (7). In spite of
identification and use of effective cancer chemopreventive
agents have become an important issue in public health-
related research, miscarriage of the intervention by some
•-) and hydrogen peroxide
intracellular ROS and
antioxidants makes necessary the evaluation of safety
before recommending use of antioxidant supplements for
chemoprevention (8). Chemopreventive agents include
indoles, catechins, pharmaceutical agents, vitamins,
organosulfur compounds (glutathione), sesquiterpenes
(celastrol), short-chain fatty
isoflavonoids (silymarin), and phenols (resveratrol,
curcumin) (8,9). While overwhelming levels and
uncontrolled/dysregulated actions of ROS lead to
deleterious effects, tighter regulation of those plays an
important role in cell signalling (2). As follow, decreased
levels of antioxidants can lead to oxidative stress,
accelerated aging and cancer (10).
Fruit and vegetable consumption has been inversely
associated with the risk of cancer, with the beneficial
effects attributed to a variety of protective antioxidants,
carotenoids and phytonutrients (11). A consensus exists
that a diet rich in fruit and vegetables is beneficial for
health in preventing coronary heart disease and some forms
of cancer. The nutrients responsible for the protective
action are not known, but vitamins, antioxidants and
flavonoids are among the likely candidates (9). Dietary
supplements are generally used to increase plasma levels of
these compounds. Similar results can be achieved also by
increasing the proportion of vegetables and fruit in the diet
Sophisticated antioxidant defenses directly and
indirectly protect the host against the damaging influence
of cytokines and oxidants (9). While antioxidant defenses
interact when a component is compromised, the nature and
extent of the defenses are influenced by dietary intake of
antioxidants, including sulfur amino acids. In animal
studies, in vivo and in vitro responses to inflammatory
stimuli are influenced by dietary intake of NAC, cysteine,
methionine, and taurine (13). Actually, every thiol-
containing molecule has been tested as a sole or
combinated supplement against several types of cancer
(Table 1).
Sulfur is the sixth most abundant macromineral
in breast milk and the third most abundant mineral based on
percentage of total body weight (6). The sulfur-
containing amino acids are cysteine, cystine, N-
acetylcysteine (NAC), homocysteine, homocystine,
methionine, and taurine. From now on (together with
ALA) we will refer to these sulphur-containing non
enzymatic antioxidants
supplementation has been indicated for athletes, children,
or patients with HIV. Organic sulfur can be used to
increase synthesis of SAM, GSH, taurine, and NAC (14).
SCNEA may also have clinical applications in the
treatment of some conditions such as depression,
fibromyalgia, arthritis, interstitial cystitis, athletic injuries,
congestive heart failure, diabetes, AIDS, and cancer (6).
The low toxicological profiles of these sulfur compounds,
combined with promising therapeutic effects, warrant
continued human clinical trails (6,14,15).
acids (lipoic acid),
as SCNEA. SCNEA
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Table 1. Cancer and human no-toxic dosage of sulphur-containing compounds
Sulfur nutrient Type of cancer
Cysteine Breast, colon
N-acetylcysteine Lung, skin, breast, colon
Homocysteine Lung
Glutathione Melanoma, colon, neuroblastoma, lung, liver, glioma, leukemia
Methionine Colorectal, breast, lung, liver, prostate
S-adenosylmethionine Colorectal, liver, pancreas, colon
Taurine Colon, lymphoma
Alpha-lipoic acid Liver, colon, lung, ovary, stomach, pancreas
1Key references are referred to type of cancer, 2reference 14, 3reference 6, 4reference 15
Thiol-containing compounds bind metals at their
-SH groups and have antioxidant features. Therefore, thiol-
containing antioxidants may be useful as a component of an
effective treatment for metal poisoning (16). Methionine
acts a precursor amino acid for glutathione which protects
the cells from oxidative damage and plays vital role in
detoxification (17). In addition, methionine has been shown
to chelate lead and remove it from tissues (16). ALA, is a
co-enzyme of pyruvate and the alpha-ketoglutarate
dehydrogenase multienzyme complex of the tricarboxylic
acid cycle, and has metal chelating, free radical scavenging,
and antioxidant-regenerating abilities (18). NAC has
antioxidant capacity to lead, including oxidative stress via
stimulating GSH synthesis, thereby
intracellular glutathione levels and scavenging ROS
(16,19). Experimental studies have shown that SCNEA
have beneficial effects against the detrimental properties of
metals. For example, oxidative stress induced in rats given
lead (2000ppm) with or without
(100mg/kg/day), NAC (800mg/kg/day), homocysteine
(25mg/kg/day), ALA (50mg/kg/day) show a significant
protective action by these sulphur-containing compounds
It has been tested the action of several SCNEA
working as free radical scavengers/antioxidants and as
protectors from photodamage induced
aminolaevulinic acid-mediated photodynamic therapy,
employing the LM2 cell line, derived from a mammary
murine adenocarcinoma (20). GSH, NAC, methionine and
cysteine are the best protectors for this treatment. GSH
protects organism against singlet oxygen. Intracellular
thiols play a role in the mechanisms of cancer treatment
modalities such as ionising radiation, chemotherapy and
hyperthermia (20). Some reports have shown that resistance
to chemotherapeutic agents may be due to elevated cellular
GSH concentrations (21). GSH may influence DNA repair,
as well as help to maintain membrane integrity via the
formation of mixed disulphides (Mates 2002). SAM is a
precursor of GSH which, unlike GSH itself, can readily
across the cell membranes. It has been shown that
supplementation of organ preservation solutions with SAM
instead of GSH has improved hydroxyl radical (•OH) and
singlet oxygen scavenging, as well as chelation of iron ions
(20). NAC increases GSH levels by providing cysteine and
it is known to prevent pathologies elicited by free radicals
and ROS (22). Methionine can be converted into SAM,
which through a series of transulphuration reactions yields
cysteine and from cysteine comes to GSH (20). The
aminothiol NAC can scavenge free radicals through
binding of the reduced sulphydryl group, either by thiol
mechanism or by enhancing GSH metabolism (22). NAC
Key references1
Dosage (mg/day)
1500-5000 2
200-1500 3
400-1000 4
600-1500 3
500-1000 3
400-1200 3
200-4000 3
20-300 3
by 5-
itself can equally act as a singlet oxygen quencher. In vivo,
NAC forms cysteine, cystine, GSH and mixed sulphides
Transsulfuration pathway has a limited tissue
distribution and is restricted to the liver, kidney, intestine,
pancreas and adrenals (14). The transsulfuration pathway is
a biochemical mechanism that links methionine metabolism
to the biosynthesis of cellular redox-controlling molecules,
like cysteine, glutathione, and taurine (Figure 1).
Deficiencies within the transsulfuration pathway induce (i)
the generation of ROS, (ii) homocysteine accumulation,
and (iii) contribute to humans pathologies like tumor
development (23). Methionine and cysteine can be used to
increase SAM, GSH, taurine, and NAC, and to promote
detoxification of xenobiotics via the sulfonation pathway
(6). Uptake of cystine by the Xc
exchanger provides the majority of cysteine, but a
significant percentage may be derived from methionine, via
a transsulfuration pathway (24). Re-feeding methionine to
diethylmaleate-treated cells incubated in the absence of
cystine or methionine resulted in a significant recovery in
glutathione (25).
The transsulfuration
homocysteine to cysteine and represents the metabolic link
between antioxidant and methylation metabolism (14). The
first and committing step in this pathway is catalyzed by
cystathionine beta-synthase (CBS), which is subject to
complex regulation, including allosteric activation by the
methyl donor, SAM (26). Methionine restriction can lead to
a >10-fold decrease in CBS protein levels. These
observations predict that under pathological conditions
where SAM levels are diminished, CBS, and therefore
glutathione levels, will be reduced. A mechanistic basis for
the coordinate changes in redox and methylation
metabolism that are a hallmark of several complex diseases
is explained by these observations (26). Under pathological
conditions with reduced SAM levels, namely human
hepatocellular carcinoma, CBS levels are diminished (26).
This decrease in CBS levels correlates with reduced
glutathione (25). Absence of methionine rather than the
presence of homocysteine was responsible for the observed
change in CBS levels (23). The transsulfuration pathway is
also blocked in some liver tumor cell lines, including
HepG2 and HuH-7 cells (26). These cells are unable to
form GSH from methionine but can synthesize large
amounts of homocysteine (23). Regulation to metabolic
changes observed in transformed liver cells in human liver
cancer, suggests therapeutic strategies that may be effective
- cystine-glutamate
pathway converts
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Thiol-non enzymatic antioxidants and cancer.
Figure 1. Transsulfuration pathway is illustrated. ACAT: acetyl-coenzyme A acetyltransferase, BHMT: betaine-homocysteine
methyltransferase, CBS: cystathione beta synthase, CDO: cysteine dioxygenase, CL: cystathione gamma lyase, CSD: cysteine
sulfinate decarboxylase, GCS: gamma glutamylcysteine synthase, GSS: glutathione synthetase, MAT: methionine
adenosyltransferase, MT: methyltransferase, PO: peroxidase, SAHH: S-adenosylhomocysteine hydrolase.
in diminishing the enhanced oxidant burden associated with
reduced transsulfuration flux (26).
The transsulfuration pathway is necessary for
cellular physiology because it connects the methionine
metabolic pathways to the generation of cysteine, GSH, and
taurine (14). The first step of transsulfuration involves the
formation of cystathionine from homocysteine and serine
(23). Once formed, cystathionine is cleaved by
cystathionine-gamma-lyase (CGL), releasing free cysteine
(24). In the presence of high cysteine levels, cystathionine
is directed into the GSH and taurine synthesis pathways
(14). Thus, cysteine levels are considered the limiting step
in liver GSH synthesis, where approximately 50% of the
cysteine used for GSH anabolism is derived from
methionine that was synthesized from the transsulfuration
pathway (26). When homocysteine levels are low, cysteine
flux through the transsulfuration pathway becomes down-
regulated to conserve homocysteine for the methionine
cycle (27).
Homocysteine can be converted into cysteine via
cystathionine through the transsulfuration pathway, an
irreversible process. Additionally, homocysteine can be
methylated back to methionine via the remethylation
pathway (14). Alternatively, homocysteine may be
remethylated to methionine with either N-5-methyl-
tetrahydrofolate or betaine as the methyl donor (15).
Remethylation is catalised by the enzyme methylene
tetrahydrofolate reductase, which uses folate and vitamin
B12 as cosubstrate (28). Clearance of homocysteine via the
transsulfuration pathway provides an endogenous route for
cysteine synthesis and represents a quantitatively
significant source of this amino acid needed for glutathione
synthesis (29). The combination of transmethylation and
remethylation pathways comprises the methionine cycle
which occurs in most cells (23). Cysteine is considered a
semi-indispensable amino acid whose availability is
dependent upon methionine intake (27). Nonetheless,
dietary cysteine can satisfy a proportion of the sulfur amino
acid requirement, the so-called cysteine-sparing effect on
dietary methionine requirement (30).
Cellular GSH homeostasis is maintained through
de novo synthesis from precursor sulfur amino acids
methionine and cysteine, regeneration from its oxidised
form glutathione disulfide (GSSG), and uptake of
extracellular intact GSH via Na+-dependent transport
systems (31). Cysteine is the rate-limiting amino acid in the
intracellular synthesis of glutathione (23). Cysteine is
derived from methionine via the transsulfuration pathway
through homocysteine (27). Methionine and homocysteine
are readily interconvertibles (25). When methionine is in
excess, homocysteine is catalyzed into cysteine via
cystathionine, which is facilitated by two pyridoxal 5′-
phosphate-dependent enzymes, cystathionine beta-synthase
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Thiol-non enzymatic antioxidants and cancer.
and gamma-cystathionase (14). When methionine is
needed, homocysteine is remethylated to methionine using
a methyl group provided by methyltetrahydrofolate; this
reaction is facilitated by methionine synthase, a vitamin
B12-dependent enzyme, or betaine-dependent methylation
(26). These interrelationships explain the positive
association between plasma concentrations of total cysteine
and total homocysteine (30). Maintaining normal GSH
concentration is essential to most tissues, especially the
intestine, which is constantly challenged by luminal toxins
and oxidants derived from the diet as well as endogenous
generated ROS. A marked depletion of GSH, induced by L-
buthionine-(S,R)-sulfoximine (BSO), was shown to result in
severe degeneration of jejunal and colonic epithelial cells in
mice and this intestinal damage seemed to be prevented by
concomitant GSH administration (14). Changes in the
extracellular cysteine/cystine redox status per se have been
shown to mediate proliferative signalling that is
independent of the intracellular GSH/GSSG in colon cancer
cells (31,32).
Cysteine is considered a semi-indispensable
amino acid whose availability is dependent upon
methionine intake (21). Cysteine is a constituent amino
acid of the tripeptide glutathione, and serves also as a
precursor for the synthesis of taurine, pyruvate, sulfate and
hydrogen sulfide (H2S) (15). Together with cystine (CySS),
its disulphide form, they constitute an important
extracellular thiol-disulphide
cysteine and cystine can be transported into cells through
distinct membrane transporters (23). Within cells, CySS
can participate in disulphide bond formation with
intracellular thiols, such as GSH, or be reduced to cysteine
(Figure 2). Conversely, intracellular cysteine can be
incorporated into proteins (32). Cysteine is usually a
nonessential amino acid in the diet of mammals because
dietary methionine is a metabolic precursor for cysteine
and, if present in an adequate amount, can completely
support the cysteine requirement (30). Regardless, the
conversion of methionine to cysteine does not occur in most
mammalian cells so that cysteine, or its disulfide form,
CySS, is needed to support individual cellular requirements
(31). CySS is the predominant form in the plasma, and
extensive research has characterized cysteine and CySS
transport systems and utilization of cysteine and CySS for
cell growth and protection against oxidative stress (23).
Inhibition of cysteine incorporation into GSH by BSO
sensitizes many cell types to anticancer therapy, and supply
of NAC and other cysteine precursors is a common
approach to enhance antioxidant defenses (31).
Previous studies have shown that cysteine
stimulated cell proliferation in various cells (32). However,
as cysteine is a precursor of GSH synthesis and the
administration of cysteine increases intracellular GSH
under physiological conditions, it is still unclear as to
whether the stimulatory effects of exogenous cysteine are
entirely mediated by intracellular GSH synthesis or
mediated, at least in part, by a glutathione-independent
mechanism (14). Moreover, because cystine can be readily
system. Nevertheless,
reduced to cysteine, the mechanism by which exogenous
CySS mediates cell proliferation is similarly unresolved. It
has been demonstrated that extracellular thiol-disulphide
redox status (cysteine-cystine balance in the medium)
regulated proliferation in the human colon cancer cell line,
CaCo-2 cells. In other study, the administered cysteine was
minimally converted to CySS in the medium, showing that
exogenous cysteine or CySS alone was as effective in
stimulating CaCo-2 cell proliferation as the combination of
the two amino acids (32). Hence, it is unlikely that
extracellular cysteine-cystine balance was a critical
determinant in modulating the stimulatory effect (23).
Accordingly, it was not possible to rule out the possibility
of a direct effect of cysteine or CySS on extracellular
membrane receptors, stating that exogenous cysteine
administration stimulated proliferative activity in GSH-
deficient CaCo-2 cells in the absence of GSH synthesis.
This mitogenic effect of exogenous cysteine could be
associated with enhanced expression of cyclin D1, and
facilitation of cell transition from G1-to-S phase (32).
A deficiency of cysteine has been shown down-regulate O6-
methylguanine DNA methyltransferase
expression and lead to increased toxicities by alkylating
agents (33). L-2-oxothiazolidine-4-carboxylic acid (OTC),
is metabolized to cysteine by 5-oxoprolinase, an essential
enzyme in the gamma-glutamyl cycle, and NAC, degraded
by cellular acylases to cysteine; both these compounds have
been demonstrated to be effective and non-toxic cysteine
delivery agents. The apparently similar influence of
methionine and cysteine can be explained by their
precursor-product relationship through the transsulfuration
pathway (34). Catabolism of GSH through the reactions in
gamma-glutamyl cycle provides a significant pool of
cellular cysteine, whose availability is critical for protein
synthesis, as well as the resynthesis of glutathione (14).
Gamma-glutamyl transpeptidase (GGT), which catalyzes
the hydrolysis of glutathione, and glutathione S-conjugates,
is the first enzymatic step in GSH catabolism (23). To
investigate whether the degradation of GSH and the
consequent cysteine production plays a role in MGMT
expression, it has been used acivicin, an irreversible
inhibitor of GGT (34). Intracellular cysteine content
significantly affects the level of MGMT gene expression,
and supports the notion of using GSH-enhancing drugs for
MGMT-targeted chemoprevention. Glutathione depletion
down-regulates MGMT activity in HT29 cells and cysteine
may up-regulate MGMT (34). Development of dietary or
synthetic compounds as potential cancer chemopreventative
agents against alkylating chemicals is highly attractive
because of their safety, low toxicity and ease of oral intake
for prolonged periods (33). Cysteine prodrugs (OTC and
NAC) increased MGMT activity in human lymphocytes
and cancer cell lines. Administration of these chemicals to
animals has been shown to result in significant increases of
cysteine and GSH levels in various tissues, including the
liver, kidney, lung and brain (34). This thiol enhancement
has been shown to prevent hepatic and other toxicities due
to acetaminophen, carcinogens, and anticancer drugs (33).
Consequently, cysteine may act as a metabolic signal for
cellular synthesis of MGMT and this raises the possible
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Thiol-non enzymatic antioxidants and cancer.
Table 2. Suggested antitumour activity of NAC in combined treatments with other anticancer drugs
Drug Cancer/tumour cells
Gingko biloba Oral cancer HSC-2 cells
Withaferin A Leukemia
Mitoxantrone Lung, leukemia
Etoposide Lymphoblastoid TK6 cells
Paclitaxel Ovarian HO8910 cells
(-)-Gossypol Squamous HNSCC cells
Cisplatin Kidney
Vitamin D3 analogs Leukemia HL60 cells
Cytosine arabinoside Leukemia P388 cells
Antimycin A Lung Calu-6 cells
Thymoquinone Colon cancer cells
Main NAC effect
Increase apoptosis
Decrease caspase-3
Increase G1-phase
Increase G1-phase
Increase colony formation
Increase apoptosis
Decrease p53
Increase differentiation
Increase apoptosis
Increase GSH
Decrease phosphorilation
6.1. N-acetylcysteine
NAC, a synthetic precursor of cysteine and
glutathione, is commonly used as a mucolytic agent and as
an antidote against acetaminophen-induced hepatotoxicity
(Figure 2). NAC has effectively blocked DNA binding by a
variety of carcinogens
acetylaminoflurorence, and cigarette smoke (21). Exposure
of NIH3T3 cells to NAC, increases the intracellular level of
GSH and thereby the elimination of ROS via the GPX
system (36). NAC inhibits cell proliferation and tumor
metastasis, increases DNA repair by protecting related
enzymes, and modulates immune responses as well as gene
expressions of a variety of tumor suppressor genes and
oncogenes. Hence, NAC has been considered to be one of
the most promising drugs for cancer chemoprevention. In
the dimethylbenzanthracene-induced
carcinogenesis model, a low dose of NAC modestly
decreased the occurrence of mammary cancers (21). In
many experimental models, NAC has antigenotoxic and
anticarcinogenic properties mediated at different stages of
the carcinogenesis process (35). NAC can equally enhance
the anchorage-independent growth of cells transformed by
activated ABL tyrosine kinases or Ras. This effect was not
dependent on loss of focal adhesion kinase activation (37).
NAC rescued cell growth that was suppressed by heat
shock protein (HSP) 90 inhibitors possibly by chemical
modification of their quinone moiety. NAC rendered
Rat1/BCR-ABL cells resistance to a Ras inhibitor
manumycin. In the absence of HSP90 inhibitors, NAC
stimulated the activation of MAP kinase in BCR-ABL-
transformed but not in the parental Rat1 cells (37). NAC
decreased IKK and IkB-a phosphorylation, and Rel-A/p65
and NF-kappaB binding, though the last two were affected
with less intensity compared to the NF-kappaB inhibitor
Dietary supplementation with a nutrient mixture
containing NAC has inhibitory potential on the
development of mouse lung tumors induced by urethane
(39). A similar nutrient mixture exhibited strong potential
as a useful anticancer therapeutic tool for skin cancer by
significantly inhibiting the incidence and tumor multiplicity
of 7,12-dimethylbenz-alpha-anthracene (DMBA)-induced
skin tumors both in vitro and in vivo (40). Additionally,
NAC has been used in combined treatments with other
anticancer drugs (Table 2). The antiproliferative effects of a
Gingko biloba leaf extract to cells from tissues of the
human oral cavity were interestingly studied. Toxicity to
the HSC-2 cells was lessened by NAC, through induction
of apoptosis (41). Induction of apoptosis in cancer cells has
Key reference
Figure 2. Cysteine (A), cistine (B), N-acetylcysteine (C),
homocysteine (D).
dietary regulation of MGMT expression in human tissues.
In contrast to GSH augmenting agents which increased
cellular MGMT, depletion of GSH reduced the MGMT
activity. While GSH may influence protein functions in
several ways including the maintenance of protein
sulfhydryls in a reduced state, other data suggested that
cysteine derived from GSH catabolism may regulate the
extent of MGMT synthesis (34).
Higher plasma total cysteine concentrations were
significantly associated with a lower risk of breast cancer
because cysteine is the precursor of glutathione, a powerful
detoxifying agent of carcinogens, which facilitates the
elimination of heavy metals, drug metabolites, and DNA-
damaging chemicals (35). As a result, cysteine or its
precursors might have the potential to be chemopreventive
against breast cancer (30). Moreover, cysteine, glutathione,
and NAC, can modulate immune responses (30). Cysteine
and cysteine derivatives inhibit both the transcription factor
nuclear factor-kappaB (NF-kappaB) that is induced under
physiological conditions by ROS and the expression of
several nuclear factor-κB-dependent genes including
interleukin-2 receptor α chain, tumor necrosis factor alpha
(TNF-alpha), the major histocompatibility complex, and c-
fos. Cysteine and cysteine derivatives also stimulate DNA
synthesis of several cycling T-cell clones and activate
cytotoxic T cells and thus play a regulatory role in T-cell-
mediated immune responses (30). Higher plasma total
cysteine concentrations were associated with lower risk of
breast cancer. An inhibitory effect of cysteine on breast
tumor initiation could be in part related to its role as the
rate-limiting amino acid in the intracellular synthesis of
glutathione (21). Plasma total cysteine levels were found
to be inversely associated with risk of cervical dysplasia
including benzo(a)pyrene,
rat mammary
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Thiol-non enzymatic antioxidants and cancer.
become the major focus of anti-cancer therapeutics
development. NAC has shown antitumour activity in
combined treatments with other anticancer drugs, including
withaferin A (42), mitoxantrone (43) and etoposide (44).
Increasing evidence indicates that ROS are also involved in
paclitaxel cytotoxicity. The cytoprotective roles of HSP27
include chaperoning cellular proteins, regulating apoptotic
signalling and modulating oxidative stress. Reduction of
HSP27 expression increased the in vitro chemosensitivity
of HO8910 ovarian cancer cells to paclitaxel and increased
paclitaxel-induced apoptosis and ROS production, although
NAC partly offset the effects of HSP27 siRNA (45). Tumor
cells with wild-type p53 and high levels of Bcl-xL are
cisplatin resistant but are induced to undergo apoptosis by
(-)-gossypol, making this a promising agent for overcoming
cisplatin resistance. As (-)-gossypol is known to undergo
oxidative metabolism in vivo, ROS generation may be
responsible for both off-target cytotoxicity and inactivation
of the drug. In agreement with this hypothesis, oxidation of
(-)-gossypol by pre-treatment with H2O2 eliminated its
activity. Combined treatment with NAC to block ROS
increased (-)-gossypol-induced cytotoxicity to tumor but
not normal cells. Furthermore, NAC increased the
induction of apoptosis, in both cisplatin sensitive and
resistant cells. Treatment with antioxidant to block ROS
prevents oxidative inactivation of (-)-gossypol and limits
off-target toxicity allowing more potent (-)-gossypol-
induced anti-tumor activity (46).
NAC also attenuated
importantly, diminished p53 activation during cisplatin
treatment. This was accompanied by the suppression of
PUMA-alpha, a p53-regulated
Concomitantly, mitochondrial cytochrome c release and
apoptosis were ameliorated. Notably, NAC when added as
part of a post-cisplatin treatment, was inhibitory to p53
activation and apoptosis (47). Apoptosis was induced with
the chemotherapy drugs VP-16 and cisplatin, in B
lymphoma cells. NAC inhibited cisplatin-induced cell
death and this is attributed to its known ability to react
directly with and inactivate cisplatin before it enters the cell
NAC significantly increased the viability of lead-
induced apoptotic HepG2 cells in a dose-dependent
manner. Interestingly, the addition of NAC to lead nitrate-
treated HepG2 cells significantly decreased cellular content
of ROS (19). 1alpha,25-dihydroxyvitamin D3 (VD3) and
the EB1089 analog are well known for their roles in the
modulation of proliferation and the differentiation of
several malignant cells.
antioxidants induced a slight increase of differentiated
HL60 cells. The highest differentiation effect occurred in
the case of the EB1089-NAC association. Thus, antioxidant
agents demonstrated a protective effect against VD3 and
EB1089 oxidative cytotoxicity and an enhancement of the
monocyte differentiation (49). Treatment for 13 weeks with
NAC of human prostatic cancer cell lines DU145 and PC3
caused inhibition of cellular proliferation, reducing ROS
levels (50). In addition, cytosine arabinoside (1-beta-d-
arabinofuranosylcytosine; Ara-C) is the most important
antimetabolite chemotherapeutic drug used for acute
•OH accumulation, and
apoptotic gene.
NAC associations with
leukemia. Interestingly, NAC, significantly blocked Ara-C-
induced cytotoxicity (51).
Homocysteine is a sulfur-containing amino acid
present in the blood and tissues but not incorporated into
protein (Figure 2). Numerous clinical studies have shown
that elevated plasma homocysteine levels are strongly
associated with increased risk for pathological status
(10,11,14). Homocysteine is a very prominent uremic
toxin. Homocysteine, formed by demethylation of
methionine, displays an antioxidant effect on cellular
systems at micromolar levels (18). As stated before,
homocysteine can be converted into cysteine via
cystathionine through the transsulfuration pathway, an
irreversible process. Homocysteine can also be methylated
back to methionine via the remethylation pathway. The
combination of transmethylation and remethylation
pathways comprises the methionine cycle which occurs in
most cells (14). Experimental evidence has indicated that
homocysteine promote vascular damage through oxidative
stress. High homocysteine levels are linked to poor
nutrition, in particular, deficient levels of folate, and
vitamins B6 and B12 (16,28). Homocysteine elevation may
result of inherited disorders of enzyme activity or from
enzyme cofactor deficiencies of cobalamin, folate, or
pyridoxine with subsequent interruption of homocysteine
metabolic pathways (15). Both in vitro and in vivo work
shows that homocysteine can activate cells to proliferate
being a potent stimulant of DNA synthesis (52). Oral
administration of
hyperhomocysteinemia leads
proliferation rates (53). Oxidative stress, altered glutathione
levels, and hyperhomocysteinemia play critical roles in
Alzheimer's disease. Hyperhomocysteinemia
correlated with increased
glutathione ratio, with decreased GST activity and
increased lipid peroxidation in mice (54).
The level of plasma homocysteine, long known
to be B vitamin dependent, has recently been shown to be
inversely associated with plasma selenium concentration in
human subjects. Although the effect of folate, PLP and
vitamin B12 concentrations on plasma homocysteine
correlated with selenium concentration, supplementation
with selenium does not affect total homocysteine
concentration. Glutamate-cysteine ligase (GCL) converts
cysteine to gamma-glutamyl cysteine (on the pathway to
GSH) and may thereby aid removal of homocysteine (25).
Men have higher plasma levels of total homocysteine than
do women, but the mechanism of this sex-dependent
difference is not known. It has been investigated regulation
by testosterone of CBS. Testosterone downregulates CBS
expression via a posttranscriptional mechanism in the
androgen-responsive prostate cancer cell line, LNCaP (29).
This diminution in CBS levels is accompanied by a
decrease in flux through the transsulfuration pathway and
by a lower intracellular GSH concentration (14).
Low millimolar concentrations of homocysteine-
induced micronuclei in L5178Y and HL60 cells but did not
to increased cellular
brain reduced/oxidized
Page 8
Thiol-non enzymatic antioxidants and cancer.
enhance the DNA damage. Homocysteine protected cells
challenged with H2O2 from oxidative stress. This was
accompanied by an increased cellular GSH level (27).
Homocysteine is a normal product in the metabolism of the
essential amino acid methionine (25). While healthy adults
have homocysteine levels lower than 12 µM, the levels of
end-stage renal disease patients frequently exceed 100 µM.
Homocysteine has often been considered a strong predictor
of both cardiovascular and non-cardiovascular mortality in
the general population and of cardiovascular mortality in
end-stage renal disease patients (27).
Additionally, hyperhomocystenemia has been
discussed as a new risk factor for cancer and as a potential
tumour marker. This is in line with studies revealing a
positive correlation between plasma homocysteine and
genomic damage (micronuclei) in healthy individuals (55).
The DNA of dialysis
hyperhomocysteinemia is often hypomethylated, which
may be due to the inhibition of methyltransferases by the
accumulation of homocysteine or the homocysteine
precursor SAM (27). If we consider that methionine-
dependent tumors produce large amounts of homocysteine
from methionine, it is plausible that homocysteine is one of
the signals required to recruit macrophages to the site of
tumor. Further studies will be needed to analyze the effect
of local hyperhomocysteinuria that has resulted from
transsulfuration deficiencies on the development of optimal
microenvironments for tumor development (23). It has been
also described homocysteine-induced genomic damage as
revealed by the micronucleus frequency in L5178Y and
HL60 cells (55). Indeed, it has been proposed that some of
the adverse effects associated with hyperhomocysteinemia
may be due to the generation of ROS (56). Homocysteine
treatment did not increase the amount of ROS in HL60
cells. This is in line with observations in HUVEC and
LLC-PK1 cells, in which homocysteine failed to elicit an
oxidative stress response. Thus, the generation of oxidative
stress by homocysteine may be cell type-specific (27). This
idea is supported by a publication stating that homocysteine
can both inhibit and promote low-density lipoprotein
oxidation depending on the experimental conditions (57).
However, it was observed a reduction of oxidative stress in
cells challenged with H2O2 (27). This reduction may be due
to the conversion of homocysteine to GSH by the
transsulfuration pathway, which would be a fast reaction
(23). It can be concluded that homocysteine can exert dual
effects in vitro: genotoxic on the one hand but antioxidant
on the other. Whether the detected antioxidant capacity of
homocysteine is relevant for any situation in patients
remains to be elucidated (Fink 2007).
Recent findings emphasize a relationship
between elevated homocysteine levels and human
hepatoma (30), colorectal cancer (58), and prostate cancer
(59). Increasing supplemental folic acid can reduce
homocysteine concentrations, which may have implications
for pathological status (10,12), including lowering cancer
risk (60). Serum homocysteine decreased significantly
throughout the course of treatment of patients with
metastatic colorectal carcinoma
combination of bevacizumab, oxaliplatin, 5-fluorouracil
patients suffering from
treated with the
and leucovorin (61). A combination of adenosine and
homocysteine potentiates
cytotoxicity, but does not modulate activation of NF-kappa
B transcription factor controlling the expression of various
TNF-alpha-inducible genes
homocysteine at concentrations that enhance TNF-alpha-
induced cytotoxicity accumulate
homocysteine (SAH), a potent inhibitor of SAM-dependent
methylation reactions (63). In addition, preloading L929
cells with SAH resulted in enhanced responses to TNF-
alpha, suggesting that SAH potentiates TNF-alpha-induced
cytotoxicity (62). Moreover, the combination of adenosine
and homocysteine changed the dependency of the TNF-
alpha-mediated cytolysis on ROS (63). In the absence of
adenosine and homocysteine
cytotoxicity was inhibited by antioxidants. In the presence
of adenosine and homocysteine, TNF-alpha-mediated
cytotoxicity is not inhibited by these antioxidants (62). A
combination of adenosine and homocysteine selectively
modulates TNF-alpha-mediated
changing the TNF-alpha-induced activation of NF-kappa B
(63). It has also been shown that adenosine and
homocysteine enhance cAMP responses. Adenosine and
homocysteine-induced modulation
cytotoxicity seems to be a general phenomenon and can be
observed in other cancer cell lines including HeLa and
WEHI cells (62). Besides oxidative stress and vascular
damage, homocysteine has a great importance in regulating
DNA methylation through SAM, the main methyl donor in
eukaryotes (23). Alterations of SAM and methylation were
evidenced in Alzheimer disease and in elderly.
Homocysteine accumulation induced through vitamin B
deprivation could impair the methylation capacity with
consequent amyloid-beta upregulation. Moreover, it was
found that homocysteine alterations had an effect on
neuroblastoma but not on glioblastoma cells in Alzheimer
disease (64).
Dehydroascorbic acid (DHA) has the remarkable
ability to eliminate the aggressive mouse tumours, L1210,
P388, Krebs sarcoma, and Ehrlich carcinoma (65). Cancer
cells (but not normal cells) contain measurable quantities of
homocysteine thiolactone (HTL). Dehydroascorbic acid
reacts with homocysteine thiolactone converting it to the
toxic compound, 3-mercaptopropionaldehyde
Rapidly-dividing tumour cells make unusually large
amounts of HTL and that administered dehydroascorbic
acid enters the cells and converts the thiolactone to
mercaptopropionaldehyde which kills the cancer cells.
HTL synthesis is mammalian cells was first demonstrated
in three malignant cell lines, human cervical carcinoma
(HeLa), mouse renal adenocarcinoma (RAG), and Chinese
hamster ovary carcinoma (CHO) (65). Homocysteine itself
does not occur in proteins and there is no enzyme for
activating it. Nonetheless, homocysteine is erroneously
activated by methionyl-tRNA synthetase in mammals. This
is recognized as an error and the homocysteinyl-AMP is
edited-out whereupon it converts to homocysteine
thiolactone (66). The presence of HTL in cancer cells can
be a finding of major interest which has not yet been fully
developed. It may be related to two conditions in rapidly-
dividing cells: (i) the high demand for methyl groups which
(62). Adenosine and
cytotoxicity without
of TNF-alpha
Page 9
Thiol-non enzymatic antioxidants and cancer.
Figure 3. Glutathione.
leads to homocysteine formation, (ii) the high rate of
protein synthesis which increases the risk of homocysteine
misactivation. Injected DHA would enter the tumour cells
where it would convert the endogenous HTL to 3-
mercaptopropionaldehyde (65). The latter compound
combines the toxicities of aldehydes and mercaptans and
would be expected to kill the tumour cells. The indicated
mechanism has obvious advantages for cancer therapy; the
toxic agent is generated inside the cancer cells and appears
to be selective for cancer cells (66). The therapeutic
potential of this mechanism might be further increased by
giving methionine or a folic acid antagonist (methotrexate)
along with DHA to increase HTL formation in cancer cells
Modulation of gene expression by oxidative
damage affects carcinogenesis by altering the epigenetic
effects and chromosomal rearrangements (23). Epigenetic
effects on gene expression stimulate growth signals and
proliferation, while chromosomal
contribute to neoplasic progression (67). In addition, a
redox imbalance is shown to stimulate protein kinase and
poly(ADP ribosylation) pathways,
transduction and promoting tumor development (23).
Interestingly, studies examining the effects of high
homocysteine levels show
polymerase (PARP) enzymes repair single strand DNA
breaks induced by genotoxic agents or oxidative stress (67).
In all related cases, the initiation potential of oxidants
seems to contribute to carcinogenesis by inducing DNA
base changes in certain oncogenes and tumor suppressor
genes (23). It is shown that the •OH is able to activate
certain oncogenes, such as K-ras and C-Raf-1 (68). Point
mutations in CpG dinucleotides are frequently found in
certain tumor suppressor genes, such as p53 and ras,
leading to inactivation (23). Furthermore, •OH attacks cells
containing mutant or absent p53, resulting in failure to
arrest in G1, and reducing cell capacity to repair damaged
DNA (67). This increase in replication errors can initiate
additional oncogene activation and tumor suppressor gene
inactivation, ultimately contributing to malignancy (23).
yl]amino]-5-oxopentanoic acid, is a very important
tripeptide present in significant concentrations in all tissues
(69). Glutathione is not an essential nutrient since it can be
synthesized from the amino acids L-cysteine, L-glutamic
acid and glycine, forming
cysteinylglycine (Figure 3). Its reduced form (GSH) and
affecting signal
that poly(ADP-ribose)
oxidized form (GSSG), which can be inter-converted by the
enzymes GPX and GR, constitute the major intracellular
thiol-disulphide redox system in mammalian cells (14).
Glutathione serves several vital functions, including
scavenging peroxides, modulating
processes (cell proliferation, apoptosis, microtubular-
related processes, and
maintaining the essential thiol status of proteins by
preventing oxidation of -SH groups or by reducing
disulphide bonds induced by oxidative stress, detoxifying
electrophiles, and providing a reservoir for cysteine (32).
Glutathione is synthesized in two sequential
ATP-dependent reactions
glutamylcysteine synthetase (GCS), the rate-limiting
enzyme, and glutathione synthetase (GSS) (23). Other
factors in the regulation of the de novo GSH synthesis are
the availability of cysteine and the concentration of GSH
itself that inhibits, by a feedback mechanism, GCS activity
(26). In the presence of oxidative stress, GSH concentration
rapidly decreases while GSSG, potentially highly cytotoxic,
increases because of the reduction of peroxides or as a
result of free radical scavenging (4). This has two important
consequences: First, the thiol redox status of the cell will
shift and activate certain oxidant response transcriptional
elements, and second, GSSG may be preferentially secreted
from the cell and degraded extracellularly, increasing the
cellular requirement for de novo GSH synthesis (4).
Glutathione disulfide can also be reduced back to GSH by
the action of GR utilizing NADPH as a reductant (3). The
high intracellular concentration of glutathione (usually 5-
10 mM) together with GPX and GR makes the molecule an
effective redox buffer to absorb excessive free radicals
(70). In mammalian cells, the cycling between GSH and
GSSG serves to remove ROS such as H2O2 produced due to
either cellular respiration or metabolism of toxic
substances, and protect cells from oxidative injuries (31).
Besides the two-step enzymatic pathway catalyzed by GCS
and GSS, cells can synthesize glutathione from a reduction
of GSSG to GSH by NADPH. Elevated tissue GSH has
been shown to prevent cancer formation (1). A method of
assessing cellular redox state was proposed by Jones (71)
basing on the Nerst equation: Eh = −252 + 30 log
([GSS]/[GSH]2). The increase in GSH level in response to
xenobiotics has been reported to be associated with an
elevation in the activity of GCS (70). It was suggested that
GSH can feedback inhibit GCS activity and a transient
decrease in GSH can release the inhibition on its activity.
While a high GSH content may be important for carrying
out detoxification, the elevated GSH level could cause a
disturbance in the balance in cellular functions (72). One of
the problems associated with a high GSH/GSSG is the
suppression of cell growth and proliferation (70).
The central role of reduced GSH appears clear in
intracellular endogenous antioxidant defenses as it is
involved in all the lines of protection against ROS (72).
Under normal cellular redox conditions, the major portion
of this regulator is in its reduced form and is distributed in
nucleus, endoplasmic reticulum and mitochondria (73).
Additionally, GSH may be covalently bound to proteins
through a process called glutathionylation and acts as a
critical cellular
immunological function),
catalyzed by gamma-
Page 10
Thiol-non enzymatic antioxidants and cancer.
coenzyme of numerous enzymes involved in cell defense
(2). Glutathione can directly scavenge free radicals or act as
a substrate for GPX and GST during the detoxification of
H2O2, lipid hydroperoxides and electrophilic compounds
(3). Glutathione peroxidases constitute a family of
enzymes, which are capable of reducing a variety of
organic and inorganic hydroperoxides to the corresponding
hydroxy compounds, utilizing GSH and/or other reducing
equivalents (4). There are several tissue-specific GPX's that
exhibit tissue-specific functions. All of them are
selenoproteins and their primary function is to counteract
oxidative attack (3). During the catalytic cycle, selenium is
oxidized by the hydroperoxides to a selenic acid derivative.
This intermediate is subsequently reduced by the electron
donor (69). When GSH is used, a seleno-disulfide is
formed, which is cleaved by a second GSH molecule to
yield the reduced GPX. During catalysis the oxidation state
of the enzyme depends on the relative concentration of the
reducing (GSH) and oxidized (hydroperoxides) substrates.
Glutathione peroxidases are ubiquitously distributed (4).
GSTs are three enzyme families (cytosolic, mitochondrial
and microsomal) that detoxify noxious electrophilic
xenobiotics, such as chemical carcinogens, environmental
pollutants and antitumor agents (69). GSTs exert those
protective effects because they are able to catalyze the
conjugation of GSH with oxidation end products and
represent a second line of defense against the highly toxic
spectrum of substances produced by ROS-mediated
reaction (4). Both GPX and GST activities can eventually
lower the level of total intracellular GSH (2). GR is a
flavoenzyme and is represented by a single-copy gene in
humans. It has been observed that exposure to agents that
lead to increased oxidative stress also leads to an increase
in its mRNA content (4). The recycling pathway for GSH
formation is fundamental in the metabolism of GSH-
dependent defense reactions (2). In conclusion, the
presence of GSH is essential, but not in itself sufficient, to
prevent the cytotoxicity of ROS, being of fundamental
importance the functionality of the glutathione-dependent
enzymes, which participate in the first and second lines of
defense (4).
Many foreign chemicals get attached to GSH,
which is really acting as a detoxifying agent (72). In fact,
alteration of GSH metabolism is one of the most essential
antioxidative defense mechanisms against oxidative
damage and cancer (69). In the reduced state, the thiol
group of cysteine is able to donate a reducing equivalent
(H+ + e-) to other unstable molecules, such as ROS (2). In
donating an electron, glutathione itself becomes reactive,
but readily reacts with another reactive glutathione to form
GSSG. Such a reaction is possible due to the relatively high
concentration of glutathione in cells (up to 5 mM in the
liver). In healthy cells and tissue, more than 90% of the
total glutathione pool is in the reduced form and less than
10% exists in the disulfide form. An increased GSSG/GSH
ratio is considered indicative of oxidative stress. For
example, breast tumor and hepatoma cell lines show altered
GSH production (73). GSH redox status is reshaped in
blood of Ehrlich ascites-bearing mice (1). This effect is
mainly due to an increase in oxidized GSH levels. Two
reasons may explain the increase in blood oxidized GSH:
(i) the increase in peroxide production, in addition to
changes affecting the GSH-related antioxidant enzyme
activities, leading to GSH oxidation within the red blood
cells; and (ii) an increase of oxidized GSH release from
different tissues into the blood. Nevertheless, GSH and
peroxide levels are higher in the cells when they proliferate
actively, like cancer cells (3). Aromatic amines, such as
benzidine, initiate bladder cancer in humans. Glutathione
inhibited transformation of benzidine by reactive nitrogen
oxygen species (RNOS) like NO+ and ONOO-. Inhibition is
at least in part due to conjugate formation because GSH is
known to react with RNOS forming S-nitrosoglutathione
Cancer cells have a high demand for cysteine to
provide GSH and drive cell growth and proliferation. C6
glioma cell line is
astrocytoma/glioblastoma cells in which to study synthesis
of glutathione. Incubation of cells with a sub-toxic
concentration of glutamate analogues (gliotoxin) leads to
an increase in the contribution of the transsulfuration
pathway to GSH synthesis because glutathione depletion
causes a JNK- and p38 mitogen activated protein kinase
(MAPK)-mediated increase in expression of CGL (24).
Notably, in the presence of both methionine and cystine,
the concentration of GSH exceeded the resting level (14).
Transsulfuration provides a compensatory mechanism that
is upregulated in response to depletion of GSH or oxidative
stress in C6 cells. These results validate the use of
gliotoxins as a tool for studying the contribution of the
transsulfuration pathway to GSH synthesis in response to
depletion of intracellular
phosphorylation takes place in the final step of the stress
activated protein kinase (SAPK) pathway that occurs in
many cells as a prelude to apoptosis (72). It has been
proposed that a depletion in GSH triggers activation of this
pathway, therefore stimulating the transsulfuration pathway
and channelling methionine towards production of cysteine
(24). It is significant that GSH depletion caused by mild
oxidative stress in hepatocytes led to a sustained increase in
JNK activity, hence sensitizing the cells to TNF-induced
apoptosis (75). Consequently, blockade of the exchanger is
viewed as a potential target for cancer therapy (24).
However, other recent evidence indicates that methionine
uptake in gliomas is high and correlates positively with
tumour viability thus questioning the potential effectiveness
of blockade of the Xc
GSH synthesis and tumour cell survival (23). Alternatively,
transsulfuration pathway functions as a pathway to ensure
continued supply of cysteine for GSH synthesis during
oxidative stress or when cystine import may be required to
drive a cystine/cysteine redox cycle in C6 glioma cells (24).
Protein kinases C (PKCs) are a family of
isoenzymes sensitive to oxidative modifications and
involved in the transduction signal pathways that regulate
cell growth (72). PKC isoforms are specifically influenced
by the amount of intracellular GSH (76). The greatest GSH
depletion is associated with a maximal ROS reduction and
accompanied by an increase in the activity of the delta
isoform (77). ROS generation induced early morphological
changes in GSH-depleted
a model system for
cysteine (24). JNK
- exchanger as a means of limiting
neuroblastoma cells
Page 11
Thiol-non enzymatic antioxidants and cancer.
characterized, at the intracellular level, by the modulation
of PKC-delta activity that was involved in the pathway
leading to apoptosis (76). These results define a novel role
of PKC-delta in the cell signaling pathway triggered by
GSH loss normally employed in the treatment of
neuroblastoma (77). GSH/GSSG ratio can be regulated by
glutathione-S-thiolation with protein cysteine residues that
may be irreversibly oxidized, and it may constitute both a
first line of defense against oxidative stress and a system
through which a redox change can be transformed into a
cellular signaling event (72). Depletion of intracellular
GSH has been reported to occur with the onset of apoptosis
in numerous studies (73) and is quite often accompanied by
an increase in ROS (3). GSH depletion modulates the
activity of specific PKC isoforms. GSH depletion induces
translocation to the mitochondria of PKC-delta (76). The
GSSG/GSH ratio is important in balancing the effects of
alkylating agents and ROS frequently produced at
membrane surfaces in response to growth factors and
cytokine stimulation (77). Once generated, ROS can alter
signaling pathways governing cell growth by specifically
altering the oxidation of protein cysteine residues that may
be reduced by GSH present at millimolar concentrations
inside cells (76). Direct oxidative injury and GSH depletion
are separate but related processes influencing the protein
oxidation state (73). Indeed, a decrease in cellular
glutathione levels consequent to the attack of free radicals
and alkylating agents enhances oxidant-induced effects
such as changes in gene expression (34). Understanding the
role of PKC-delta in the induction of early morphological
changes induced by diminished GSH levels accompanied
by the formation of oxyradicals might be useful in the
commitment of neuroblastoma cells to apoptosis and in the
clinical approach to brain cancers and their complications
There is considerable evidence both in animal
models and in human studies that alcohol-induces hepatic
injury involves GSH depletion and consequent oxidative
stress (72). Ethanol ingestion decreased glutathione levels
in the plasma, lung tissue, and lung lavage fluid, and
increased oxidized glutathione levels in the lung lavage
fluid (78). Furthermore, ethanol ingestion decreased cell
glutathione content by 95%, and decreased cell viability, in
vitro. Finally, treatment with the glutathione precursors
SAM and NAC during ethanol ingestion significantly
reduced lung damage (79).
hydrocarbons increases GSH content as a defense
mechanism against oxidative stress as well as to promote
its detoxification in the human hepatoma cells HepG2. The
increase in intracellular GSH level was due to activation of
GCS activities (70).
Many cancer therapeutic drugs are apoptosis
inducers as they can disrupt the redox balance by depleting
the intracellular thiol buffer system through extrusion or
redistribution of GSH (73). Redox regulation is an
important component of malignant cell survival. Tipping
the cellular redox balance through pharmacologic
regulation in favor of increasing intracellular ROS and/or
depleting protective reducing metabolites (such as
glutathione) may lead to oxidative stress and resultant
Polycyclic aromatic
induction of apoptosis for the treatment of cancer (80).
Deplete intracellular GSH leads to a depletion of protein
thiols as well as enhancement of ROS which eventually
leads to apoptotic cell death (81). Lower blood levels of
glutathione were also observed among patients with
cervical dysplasia or invasive cancer (78).
8.1. Glutathione in the glutamine/glutamate network
Eventually, glutamine via glutamate, is one of the
precursors for the synthesis of GSH, the major endogenous
antioxidant in mammalian cells, which protects them from
oxidative injury and cell death (3). Cancer cells have higher
GSH levels than the surrounding normal cells, which
attributes to a higher rate of cell proliferation and resistance
to chemotherapy (1). Therefore, selective tumour depletion
of GSH presents a promising strategy in cancer treatment.
Experimental studies have associated decreased GSH levels
with inhibition of proliferation and stimulation of apoptosis
(82). Glutamine metabolism is another target for alteration
in cancer development. Both glutamine uptake and the rate
of glutaminolysis (i.e., catabolism of glutamine to generate
ATP and lactate in the mitochondria) are known to increase
in tumors (83). In glutamine metabolism, mitochondrial
glutaminase (GLS) is central in the conversion of glutamine
to glutamate (3). Glutamate participates in regulation of
mitochondrial bioenergetics in many normal and cancer
cells via the tricarboxylic acid cycle for ATP production as
well as antioxidant defense through GSH synthesis (83).
The human glutaminase 2 gene (GLS2) encodes a
mitochondrial glutaminase that catalyzes the hydrolysis of
glutamine to glutamate (83). P53-inducible GLS2 regulates
intracellular glutamine metabolism and ROS levels and
promotes antioxidant defense through controlling the
GSH/GSSG ratio, although it exists the additional
possibility that regeneration of GSH from GSSG is
increased by GLS2 expression (84). Other results show
how modified Ehrlich ascites tumour cells, expressing
antisense mRNA for GLS2, contain lower levels of GSH
than normal ascites cells. In this context, these cells contain
a higher number of apoptotic cells and are more sensitive to
both methotrexate and H2O2 toxicity than normal cells.
Taken together, these results provide insights into the role
of GSH in cancer development by demonstrating that GSH
levels can alter apoptosis and tumorigenic capacity (1).
GLS2 is a unique p53 target gene to mediate the
role of p53 in both cellular energy metabolism and
antioxidant defense mechanisms (85). P53 increases GLS2
expression under both nonstressed and stressed conditions,
which results in enhanced mitochondrial respiration and
ATP generation and, furthermore, increased glutathione
levels and decreased ROS levels in cells, which in turn
protects cells from oxidative stress-induced apoptosis (85).
Furthermore, GLS2 expression is lost or greatly decreased
in hepatocellular carcinomas and the overexpression of
GLS2 greatly reduced tumor cell colony formation (84).
These results demonstrated that as a unique p53 target
gene, GLS2 is a mediator of p53's role in energy
metabolism and antioxidant defense, which can contribute
to its role in tumor suppression (85). Glutamine must be
converted to glutamate by glutaminase before entering the
TCA cycle: cancer cells that express the Myc oncogene
overexpress glutaminase and are killed by glutamine
Page 12
Thiol-non enzymatic antioxidants and cancer.
withdrawal (83). However first-generation glutamine
antagonists such as 6-diazo-5-oxo-L-norleucine and
acivicin are neurotoxic, safe glutaminase inhibitors are
under development (86).
Malignant glioma cells kill surrounding neurons
in the brain specifically by secreting the amino acid
glutamate, an obligatory waste product of glutathione
synthesis (87). Glutamate release is a plausible mechanism
that may account for the pathologic changes in bone
metastasis, since bone, like brain, is also highly sensitive to
glutamatergic disruption (88). Glutamate is released from
cancer cells as a side effect of cellular oxidative stress
protection, and that this process may be common to a
variety of cancers (83). Many cancer cells must sustain
significantly upregulated antioxidant defence mechanisms
to survive, progress, and potentially metastasize (72). To
generate sufficient GSH, an abundant source of
intracellular cysteine is required (14). Since cysteine is
rapidly oxidized to cystine in the extracellular environment,
an amino acid transport system is necessary to move the
oxidized form into the cell for a readily available cellular
waste metabolite, glutamate (88). Although the released
glutamate can act as an autocrine/paracrine signal to
encourage cell invasion, the functional consequences to the
host organ are for far greater significance for glioma
patients (88). As neurons do not have the enzymatic
capacity to effectively terminate the glutamate signal, this
excess glutamate will cause hyperexcitation, eventually
leading to neuronal death (88). While glutamate release has
been suggested as providing an adaptive advantage for
glioma cells, the host pathology may simply be a side effect
of the interaction between the cancer´s requirements for
high levels of GSH biosynthesis and the brain´s inherent
sensitivity to glutamate (88).
8.2. Glutathione and drug resistance of cancer cells
GSH content of cancer cells is particularly
relevant in regulating mutagenic mechanisms, DNA
synthesis, growth, and multidrug and radiation resistance
(73). In malignant tumors, as compared with normal
tissues, that resistance associates in most cases with higher
GSH levels within these cancer cells (21). Hence,
approaches to cancer treatment based on modulation of
GSH should control possible growth-associated changes in
GSH content and synthesis in these cells. Recent works
show that a high percentage of metastatic cells with high
GSH levels survive the combined nitrosative and oxidative
stresses. GGT overexpression and an inter-organ flow of
GSH, by increasing cysteine availability for tumor GSH
synthesis, function in combination as a metastatic-growth
promoting mechanism. Experimental evidence shows that
acceleration of GSH efflux facilitates selective GSH
depletion in metastatic cells, and sensitization of metastatic
cells to therapy (73). Efflux of GSH from melanoma cells
may facilitate metastatic
endothelium-induced oxidative/nitrosative stress. GSH
efflux was abolished in melanoma cells incubated with
methionine (likely related to a methionine-sensitive GSH
carrier previously detected in hepatocytes). GSH depletion
in metastatic cells can be achieved by using Bcl-2 antisense
cell resistance against
oligodeoxynucleotide, in combination with inhibition of
GGT, which limits GSH synthesis by preventing cysteine
generation from extracellular GSH (21).
Drug-resistant cells have the inherent ability to
maintain physiological GSH, and cellular redox state and
maintain increased cellular resistance to DNA damage (72).
GSH loss was followed by a rapid increase in ROS levels
(89). The resistant, but not drug-sensitive cells normalized
the intracellular GSH concentration along with ROS levels
and survived drug treatment (1). GSH levels are elevated in
many types of tumor cells that show increased resistance to
chemotherapeutic agents (89). GSH levels vary over a
range of 0.5-10 mM, depending on the cell type, and play a
role in the detoxification of a variety of electrophilic
compounds and peroxides via catalysis by GST and GPX.
GSH levels are rapidly depleted during apoptosis, and this
is considered a biochemical hallmark of oxidant-induced,
programmed cell death (31). Maintaining intracellular GSH
levels by either GSH precursors (such as NAC) or
inhibitors of GSH efflux can repress or delay apoptosis.
Drug-resistant cancer cells, whether derived directly from
patients or maintained in culture, have high GSH levels and
show increased GCS activity (89). GCS, along with GSS is
involved in the de novo synthesis of GSH (14). Low
molecular weight, non-enzymatic antioxidants (e.g.,
SCNEA), protect cells from ROS-mediated damage or
pathological signaling (9).
H2O2 and other peroxides are mostly eliminated
by GPX, which utilizes GSH as an electron donor in the
reduction reaction, and produces GSSG as an end product
(3). GSH was extruded equally from the sensitive and
resistant cells via methionine- or cysteine-inhibitable GSH
transporters after drug treatment (89). This GSH loss
caused a subsequent increase in ROS levels in many cell
types (1). Restoration of GSH levels in drug-resistant cells
consistent with the high GSH levels in tumor cells
possessing increased resistance to platinum-containing
compounds, alkylating agents, anthracyclines, and arsenic
trioxide (89). GSH hinders the arsenic trioxide apoptosis
induction through ROS-dependent survivin downregulation
in human lung cancer cells. Survivin is a member of the
inhibitor of apoptosis protein family. It is modulated by
ROS in several types of neoplasms including leukemias
(90). Drug-resistant cells possess high GCS activity. GSH
extrusion was significantly inhibited by methionine or
cysteine, suggesting that GSH loss occurs via a specific
transporter (89). The mitochondrial GSH level is dependent
on the cytosolic GSH concentration, and so it is possible
that a decrease in the GSH concentration impairs the GSH-
dependent ROS detoxification system (primarily GPX and
GR) in mitochondria, leading to an excess of ROS that is
released into the cytoplasm (3). Resistance to commonly
used alkylating or platinum chemotherapeutic agents is
multifactorial, involving altered drug transport along with
enhanced drug biodegradation, detoxification, and DNA
repair (89). Increased GSH levels due to an enhanced
gamma-GCS activity were reported in several drug-
resistant tumor cell lines, and in tumor cell cultures isolated
from patients whose tumors were clinically resistant to drug
therapy (89).
Page 13
Thiol-non enzymatic antioxidants and cancer.
Assuming that cysteine levels are maintained by
the breakdown of GSH, there is a shift in the redox balance
following the induction of oxidative stress, such that the
ratio of GSH to GSSG is altered in favor of GSSG (14).
GSH acts as a reducing agent, is involved in the
metabolism of xenobiotics, is a free-radical scavenger, aids
in cell-cycle regulation
mechanisms, regulates Ca2+ homeostasis, regulates protein
function and gene expression via thiol-disulfide exchange
reactions, and participates in the mitochondrial mechanisms
that link the opening of the permeability transition pore
complex to the activation of cell death (23). Different types
of tumor, including melanoma, bladder carcinoma, lung
cancer, colon cancer, and breast tumors that are multidrug
and radiation resistant, are also found to have a high GSH
content (89). Considering the homeostatic redox buffer
function of GSH and its role in inactivating some
carcinogens and protecting cells against DNA-damaging
free radicals and lipid peroxidation, it is plausible that
tumor cells may need more GSH for their survival than
other cell types (23). GST is shown to be associated with
multidrug resistance of tumor cells (31). High GSH
concentrations in cancer cells, induction of GSH in murine
melanocytes following c-H-ras
transformation, or impairment of metastatic spread by GSH
depletion have been described, further highlighting the
importance of GSH pathways in tumor development and
growth (23). Under metastatic conditions, high levels of
GSH can support a rapid cell cycle, an elevated rate of
DNA synthesis, and a block in apoptosis (3). Therefore,
GSH can be characterized as a double edged sword,
protecting non-tumor cells against oxidative stress induced
by metabolism or exogenous compounds and at the same
time, protecting tumor cells from apoptosis and
chemotherapeutic treatments, thus furthering tumor
development and metastasis (23).
Free radical-induced cytotoxicity may initiate
carcinogenesis by depleting the normal cell population and
promoting the clonal expansion of more resistant cells (23).
Glutathione depletion is widely used to sensitize cells to
anticancer treatment inducing
programmed cell death and overcoming chemoresistance
(1). It has been reported that neuroblastoma cells with
MYCN amplification are unable to start TRAIL-dependent
death and MYCN, in concert with cytotoxic drugs,
efficiently induces the mitochondrial pathway of apoptosis
through oxidative mechanisms (77). These results describe
a novel pathway of apoptosis dependent on ROS formation
and PKC-delta activation and independent of p53, Bcl-2,
and Bax levels (76). GSH loss induced by BSO leads to
overproduction of ROS and triggers apoptosis of MYCN-
amplified neuroblastoma cells (77). BSO is a GSH-
depleting agent that inhibits GCS, which is often up-
regulated in chemotherapy-resistant tumors (81). GSH
depletion is responsible for DNA damage and apoptosis in
MYCN-amplified NB cells in consequence of ROS
overproduction and activation of PKC-delta (77).
Retinoid N-(4-hydroxyphenyl) retinamide (4-
HPR) induces the overproduction of ROS in human
leukemia cells (91). 4-HPR generated large quantities of
and microtubular-related
the progression of
ROS in cell lines which expressed low glutathione levels,
these cells being the most sensitive to the retinoid (92). The
sensitivity of leukemia cells to 4-HPR could be modulated,
either by increasing intracellular glutathione contents using
all-trans retinoic acid, or by decreasing it using DL-BSO
(91). All-trans-retinoic acid increased the level of
expression of tissue transglutaminase, whereas inhibition of
this enzyme led to enhanced apoptosis (93). Our findings
indicate that the glutathione content contributes to
determining the sensitivity of cells to 4-HPR and points to
the potential application of glutathione-inhibiting agents as
enhancers in 4-HPR-based therapies (91). The cytotoxicity
of 4-HPR was analyzed in four other leukemia/lymphoma
lines (CCRF-HSB2, Molt-4, KG-1, HL-60). Cytotoxicity of
4-HPR correlated with the amount of ROS produced in
many cell lines (92). The intracellular glutathione level
varied among all the assayed cell lines, the highest levels
occurring in Molt-4 and KG-1, which were less sensitive to
4-HPR. Suppression of glutathione by BSO enhanced the
level of 4-HPR-induced ROS production and apoptosis in
Molt-4 (91).
Glutathione pathway was also altered in DND-
1A, HeLa (94) and HSC-2 cancer cell lines (41). Recovery
of GSH depletion and the reduction of O2
lung cancer cells were accompanied by the inhibition of
apoptosis (95). Data provide strong support for the
hypothesis that variation in GSH is associated with
prognosis of some types of cancer (96). Of note, GSH
contents and the activities of antioxidant enzymes such as
GR, and GST were adversely reduced by cytotoxic and
genotoxic agents on human lung carcinoma pulmonary type
II-like epithelium cell (A-549 cells) (97). It has been
reported that the intracellular GSH content has a decisive
effect on anticancer drug-induced apoptosis, which
indicates that apoptotic effects are inversely comparative to
GSH content (98). Hence, effects of tamoxifen were
prevented when mitochondrial nitric oxide synthase was
inhibited and GSH was supplemented (99). Depletion of
GSH sensitizes follicles and granulose cells to toxicant-
induced apoptosis and that supplementation of GSH is
protective. Suppression of GSH synthesis reversed
resistance to radiation as GSH synthesis ameliorates
various sequelae associated with exposure to oxidative
stress and irradiation (100).
Methionine is an essential amino acid that
influences cellular metabolism (Figure 4). Methionine
metabolism can be divided into five major but
interdependent biochemical pathways: the methionine
salvage pathway, the methionine cycle, the transsulfuration
pathway, the GSH synthesis pathway, and the taurine
synthesis pathway (23). Methionine metabolites are
essential for phosphatidyl choline regeneration in the
cytoplasmic membrane, and methionine is required for the
metabolic pathways that regulate gene expression,
chromatin structure, transcription, post-transcriptional
processing, and protein synthesis (101). Deficiencies are
responsible for many kinds of tumors, including non-small
cell lung cancer, leukemia, glioma, rectal adenocarcinoma,
•- levels in Calu-6
Page 14
Thiol-non enzymatic antioxidants and cancer.
Figure 4. Methionine (A), S-adenosylmethionine (B).
and melanoma (102). The methionine cycle is equally
responsible for (i) converting betaine into dimethyl-glicine,
which regenerates methionine from cysteine in a reaction
catalyzed by betaine homocysteine methyltransferase, and
(ii) converting 5-methylenetetrahydrofolate (5-THF) into
tetrahydrofolate (THF) by methionine synthase, which
produces methionine from homocysteine (102). It is
demonstrated that approximately 50% of all tumors are
incapable of proliferating when methionine is replaced by
homocysteine, resulting in cell cycle arrest, and eventual
death (103).
Methionine is transmethylated intracellularly to
homocysteine via SAM, the principal biological methyl
donor in mammalian cells and a precursor for polyamine
synthesis (102). Dietary factors on cancer risk include
methionine supply (Table 2), that may interact with some
genes associated with breast, lung and colorectal cancer
(17). Mitochondrial generation of ROS is increased in mice
with fatty livers induced by methionine-deficient diets
(104). Both nuclear and mitochondrial DNA are targets for
ROS-induced damage and accumulate hydroxylated bases,
that introduce mutations that promote cancer as well as cell
death (73). Fatty livers are unusually vulnerable to damage
from oxidative stress and also exhibit an increased risk for
hepatocellular carcinoma (105). Restriction of methionine
modulates Raf and Akt survival pathways and affects the
function of mitochondria in DU145 and PC3, two human
androgen-independent prostate cancer cell lines (104). This
restriction inhibits energy production (ATP synthesis) and
induces generation of ROS. Restriction of methionine in
DU145 and PC3 reduces mitochondrial membrane potential
and induces release of cytochrome c from mitochondria and
caspase-independent apoptosis
methionine restriction reduces activity of Akt, activates
caspase-9, decreases mitochondrial
phosphorylated Raf and apoptosis inducing factor, and
increases mitochondrial distribution of Bak (104). The
inhibition of Akt activity alone does not explain the ability
of methionine restriction to induce apoptosis of PC3 (106).
In fact, mitochondrial Bcl-xL is also decreased in
methionine-restricted cells (107). Under methionine
restriction, reduced mitochondrial Raf does not inactivate
(106). In DU145,
distribution of
the pro-apoptotic function of Bak. Conversely, apoptosis
induced by methionine restriction was not associated with
alterations in intracellular distribution of Raf, Bcl-2 family
proteins, or apoptosis inducing factor (104).
Methionine works as a micronutrient protective
agent involved in DNA methylation, synthesis, and repair.
Methionine shows significant protection for rectal cancer in
second and third quintiles of consumption but not for colon
cancer (108). Some risk factors for breast cancer are
dependent upon CYP1A1 genotype. Low intakes of
methionine appear to increase the risk of breast cancer in
individuals with specific genotypes (109). It is likely that
the elevated production of homocysteine by methionine-
dependent cancer cells is an adaptation that has allowed
tumor cells to survive and colonize a constantly changing,
biological environment (23). Cisplatin is an anti-neoplastic
drug extensively used in cases of head and neck cancer that
induces ototoxicity. Antioxidant drug D-methionine (300
mg/kg) can partially protect from cisplatin ototoxicity
(110). Sulphurs containing antioxidants have the potential
to compensate the side-effects of cisplatin, and it has been
observed that both isomers of methionine block the
cisplatin toxic effects in the ear and in the kidney (111). On
the other hand, intraperitoneal administration of rats within
a select dose range of seleno-L-methionine reduces
cyclophosphamide-induced toxicity (myelosuppression) on
blood cells and bone marrow (112).
9.1. S-adenosylmethionine
SAM is the major biological methyl donor for
other molecules such as DNA and plays an important
allosteric role in the regulation of sulfur metabolism (23).
Reduced SAM concentrations, as a consequence of low
methionine intake or folate deficiency, mainly lead to a
deregulation in DNA methylation and are implicated in
various cancers, including colorectal cancer (Figure 4).
Decreased methylation of DNA may contribute to loss of
the normal controls on proto-oncogene expression (14). In
humans, hypomethylation of DNA has been observed in
colorectal cancers (113).
methylation abnormalities, observed during progression of
human neoplasias, may be influenced by certain dietary
factors (114). Low dietary folate and methionine may
reduce levels of SAM (14). High dietary folate was
inversely associated with risk of colorectal adenoma, and
methionine intake was inversely associated with risk of
adenomas (113).
Under normal circumstances, most cellular
oxygen is consumed in the cytochrome c oxidase complex
of the respiratory chain, a system that does not generate
ROS (2). However, within the respiratory chain, the
ubiquinone pool of complex III of respiration generates O2
as a result of single electron transfer to molecular oxygen
(115). Ethanol metabolism leads to the generation of
acetaldehyde and free radicals that bind rapidly to
numerous cellular targets, including components of cell
signaling pathways and DNA (116). In addition to direct
DNA damage, acetaldehyde depletes GSH (115). Chronic
ethanol abuse leads to induction of hepatocyte microsomal
cytochrome P450 2E1, an enzyme that metabolizes ethanol
Accumulation of DNA
Page 15
Thiol-non enzymatic antioxidants and cancer.
Figure 5. Taurine (A), alpha-lipoic acid (B).
to acetaldehyde and causes further free radical
production and aberrant cell function leading to cancer
development (116). SAM is employed as dietary
antioxidants for therapeutic purposes. SAM has generated
considerable interest as an antioxidant for use in protecting
against the deleterious effects of ethanol on the liver (115).
The role of SAM in transsulfuration is central as a
precursor in GSH synthesis, GSH representing a main
hepatocellular antioxidant (25). Despite this, the role of
SAM as an exogenous antioxidant therapy remains
controversial (115).
SAM exhibits a chemoprotective effect (54).
Likely this effect is mediated by counteracting, oxidative
stress and NF-kappaB activation. To test this hypothesis
F344 rats were subjected to hepatocarcinogenesis with or
without SAM (38). Lipid-peroxidation was decreased by
antioxidants, but only SAM increased glutathione. SAM, in
its regulation from IKK downwards, abolished the NF-
kappaB activation. Although all antioxidants inhibited
oxidative stress as shown by reduction of lipid
peroxidation, not all exerted the same effect on NF-kappaB
signalling pathway and only SAM increased GSH. The
mechanisms exerted by SAM makes this compound a
potential liver cancer therapeutic agent (38). SAM
potentiated SOD and GST activity and restored altered
brain glutathione and erythrocytes lipid peroxidation. These
results underline the importance of SAM as neuroprotective
compound, acting both on methylation and oxidation
metabolism (54).
Accumulation of genetic changes characterizes
the progression of cells, initiated by carcinogens, to full
malignancy (102). A decrease in SAM content in the liver,
associated with DNA hypomethylation in rat liver, during
the development of preneoplastic foci, and in neoplastic
nodules and hepatocellular
Reconstitution of the methyl donor level in the liver by
SAM administration inhibits growth and induces
phenotypic reversion and apoptosis of preneoplastic cells
(102). A 6-month SAM treatment results in a sharp and
persistent decrease in development of neoplastic nodules,
suggesting a long duration of SAM chemopreventive effect.
SAM treatment results in overall DNA methylation and
partial methylation of c-Ha-ras, c-Ki-ras, and c-myc (103).
SAM is a labile molecule that, at physiologic
temperature and pH, is spontaneously cleaved into 5′-
carcinomas (103).
methylthioadenosine (MTA) and homoserine lactone (117).
Treatment of rats with exogenous SAM results in recovery
of SAM level in preneoplastic liver cells, as well as in a
partial reconstitution of the MTA pool, which is greatly
reduced in preneoplastic cells. Administration of MTA to
rats induces a dose-dependent inhibition of the
development of preneoplastic liver lesions, associated with
inhibition of DNA synthesis and increase in remodeling of
preneoplastic lesions. It was considered that the SAM
chemopreventive effect might depend, at least partially, on
its transformation into MTA
chemopreventive effect is associated with a marked dose-
dependent inhibition of ornithine decarboxylase activity, a
key enzyme in polyamine synthesis (118). Results of recent
research on the protective effect of SAM and MTA on
oxidative liver damage by carbon tetrachloride have shown
an antioxidative effect of both molecules. There is evidence
that the two molecules exert their effect by different
mechanisms: maintenance of a high GSH pool occurs in
carbon tetrachloride-intoxicated rats, as a consequence of
SAM treatment, whereas MTA, being a thioether
compound, may exert a direct antioxidant effect, even
without affecting GSH pool. The question arises whether
the therapeutic use of SAM is preferable to that of the more
stable MTA molecule. In human beings, dysplastic and
regenerative liver nodules and neoplastic nodules are
preneoplastic lesions. At present, it is easier to envision a
therapeutic use of SAM, as a chemopreventive agent,
because of its current use in human beings for treatment of
liver injury (103). SAM and MTA exert a proapoptotic
effect in colon cancer HT-29 and RKO cells. HT-29 and
RKO differ in p53 status: HT-29 cells express inactive p53,
whereas RKO cells express wild-type p53. Hence, the
proapoptotic effects of SAM and MTA are independent of
p53. Although SAM and MTA are proapoptotic in colon
cancer cells, they have no toxic effects in NCM460 cells, a
normal colon epithelial cell line (117). In contrast to liver
cancer cells, SAM and MTA had no effect on Bcl-xS
expression in colon cancer cells (119). In conclusion, SAM
and MTA are proapoptotic in colon cancer cells but not
normal colon epithelial cells. The evidence that SAM is
non-toxic to normal colon epithelial cells is consistent with
its excellent safety profile and lack of significant side
effects. Thus, SAM and MTA may be attractive agents in
the chemoprevention and in the treatment of colonic
neoplasia (117).
Taurine (2-aminoethanesulphonic acid) is the
most abundant intracellular amino acid in humans, being a
non-essential sulfur-containing
membrane-stabilizing and cytoprotective properties (99).
Taurine is a free amino acid in inflammatory cells, where it
is thought to be cytoprotective (Figure 5). Therapeutically,
taurine possesses antidiabetic, antihypertensive, antioxidant
and hepatoprotective activities (120). One of the possible
mechanisms of its pharmacological action involves the free
radical scavenger activity. Taurine treatment showed
reduced lipid peroxidation and protein oxidation (measured
as protein carbonyl content) in the liver mitochondria (99).
Taurine is a conditionally essential non-proteinogenic
(103). The MTA
β-amino acid with


text:
Amid some of the most well-orchestrated and well-attended protests from parents and activists in recent history, the School Reform Commission last week voted to close down nearly two-dozen schools and consolidate a dozen others.
Amid some of the most well-orchestrated and well-attended protests from parents and activists in recent history, the School Reform Commission last week voted to close down nearly two-dozen schools and consolidate a dozen others.
Hundreds of North Side residents are asking their alderman, Joe Moore, to publicly oppose the opening of future charter schools in Chicago's 49th ward.
Last summer, while some of their peers were at home playing video games, 20 Pflugerville ISD students were learning how to design them.
A program in Boise schools is spreading, and providing students with a better chance at graduating and going on to college.
The thought is that the city of Memphis will come up with some amount of funding for the consolidated school district once the schools consolidation crosses over into the new fiscal year that starts July 1.
If you're itching to tell Portland Public Schools about your educational priorities this month, you're in luck.
As a free tool, SymbalooEDU, will help Spanish educators stay current with technology development in education while also helping them to survive the current budget cuts in Spain.
The new SR-series access switches combine cloud-management, on-demand provisioning and secure branch routing to reduce the complexity of today's "mobile first" enterprise.
Enterasys Networks, a Siemens Enterprise Communications Company, will be exhibiting and presenting sessions on BYOD, network development and Web filtering for K-12 classrooms at BrainStorm 14.0.
On the Kobo website, readers will now find some of ASCD's bestsellers by notable education authors, including: Charlotte Danielson's Enhancing Professional Practice, 2nd Edition; Eric Jensen's Teaching with Poverty in Mind; and Carol Ann Tomlinson's How to Differentiate Instruction in Mixed-Ability Classrooms, 2nd Edition.
On Monday Governor Rick Scott delivered a check for $15 million to the cash strapped school district as part of the state's School Recognition Program which gives financial rewards to schools based on sustained or significantly improved student achievement in reading, mathematics, science, and writing.
Carroll County in east central Ohio is at the center of the oil and gas drilling boom, and the local school district wants to tap into that boom – literally.
Last month, Gov. John Kasich made waves with his school-funding proposal. The waves have not ceased ... and local districts are still bobbing up and down in rather deep troughs.
Built for the Common Core, i-Ready Diagnostic & Instruction combines a valid and reliable growth measure and individualized instruction in a single online product. To reach one million assessments in less than two years is a testament to the fact that i-Ready delivers a much-needed resource for educators across the country. The 1 millionth assessment was completed by two different students, in two different states at the exact same time on February 26 at 1:55 p.m. EST.
A new e-book, Social Media in Education: Enhancing Learning and Managing eSafety, from Bloxx, addresses the challenges schools face in harnessing social media information and collaborative learning tools, while protecting students from inappropriate content and dangerous malware.

subset: commoncrawl


text:
Bills QB, coach differ on offense's performance
PITTSFORD, N.Y. -- Bills quarterback EJ Manuel and coach Doug Marrone had widely conflicting assessments on how the offense performed on the third day of training camp.
Despite numerous errant throws, Manuel didn't see much of anything go wrong Tuesday.
"Nah, good day," he said. "I thought we ran the ball well. I thought we threw the ball well."
Marrone saw things differently. He agreed when asked if the session was sloppy. And Marrone then provided one-word answers over what he didn't like.
"Execution," he said. "Communication."
Marrone also wasn't pleased with Manuel's inability to get the ball off during several 7-on-7 drills, when the quarterback decided to take off and run.
"I'm not fine with that," Marrone said. "I mean, it's 7-on-7. We want to throw the football down the field."
Marrone did credit Manuel for at least not forcing throws and risking interceptions.
Aside from rookie receiver Sammy Watkins making a few more highlight-reel catches, not much else went right for the Bills' passing game on the first day players wore pads.
"We're not even close to where we'd like to be," Marrone said. "We've got a lot of work ahead of us, as you guys have witnessed today."
Manuel's best throw came on his first pass in 7-on-7, when he hit a wide open Watkins in stride up the right sideline. Watkins, drafted fourth overall, also made a leaping grab at the sideline of a very wobbly pass thrown by Manuel.
Those were among the few highlights.
During one four-snap set, Manuel pulled up and ran twice, and then threw two incompletions. And he had a tough time getting in sync with second-year receiver Robert Woods.
On one play, Woods made a quick cut only to have Manuel sail the ball well over his head. On another, Woods went deep, only to have Manuel throw short.
The Bills' offense, and Manuel in particular, are under plenty of scrutiny this summer after struggling for much of last season. As a rookie starter, Manuel's development was slowed by three separate knee injuries that limited the first-round pick to 10 games.
Manuel still struggled when he was healthy. He had a 4-6 record with 11 touchdowns, nine interceptions and a 58.8 completion percentage that ranked him 13th among AFC quarterbacks with more than 300 attempts.
The Bills invested plenty this offseason around Manuel. They gave up next year's first-round draft pick to move up five spots to select Watkins and used three of their remaining six selections on offensive linemen. Buffalo also added size at receiver by acquiring Mike Williams in a trade with Tampa Bay.
General manager Doug Whaley said the moves were made with an eye on helping Manuel progress.
"It's tough as a rookie," Whaley said. "But by the time we leave training camp, you should see a guy that's standing tall in the huddle and quick and decisive with his decision-making."
So far, Manuel has again looked inconsistent.
He's had difficulty with his accuracy in completing short passes across the middle. He's been hesitant in the pocket. And it hasn't helped that the patchwork offense line -- minus left tackle Cordy Glenn (illness) -- is having difficulty handling the Bills well-established pass-rushers.
Marrone provided no excuses for the offense's struggles.
"I want them to perform better, no doubt about it," he said.
NOTES: DT Marcell Dareus was expected to arrive at camp Tuesday after his flight from Alabama on Monday night was delayed by bad weather. Dareus was excused from camp to attend a court hearing, where he agreed to enter an NFL substance abuse program to have two felony drug charges dismissed. ... The Bills signed free agent LB Xavius Boyd and released LB Darrin Kitchens by designating him waived/injured. Kitchens hurt his left leg in practice Monday. ... DB Brandon Smith (left knee) is out indefinitely after he was also hurt Monday. ... The Bills practice Wednesday morning before getting a day off.
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Copyright 2014 by The Associated Press
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