Antiporter: A Potential Target
for Therapy of Cancer and
MAISIE LO,1,2YU-ZHUO WANG,3,4AND PETER W. GOUT3*
1Department of Experimental Medicine, University of British Columbia, Vancouver, BC, Canada
2Department of Cancer Genetics, British Columbia Cancer Agency, Vancouver, BC, Canada
3Department of Cancer Endocrinology, British Columbia Cancer Agency, Vancouver, BC, Canada
4The Prostate Centre at Vancouver General Hospital, Vancouver, BC, Canada
glutamate. Its main functions in the body are mediation of cellular cystine uptake for synthesis of glutathione essential for cellular
a role in certain CNS and eye diseases. This review focuses on the expression and function of the x?
particular emphasis on its role in disease pathogenesis. The potential use of x?
and/or sensitizing cancers is discussed.
J. Cell. Physiol. 215: 593–602, 2008. ? 2008 Wiley-Liss, Inc.
ctransporter in cells and tissues with
cinhibitors (e.g., sulfasalazine) for arresting tumor growth
Plasma membrane amino acid transporters allow regulated
bidirectional transfer of specific amino acids across the plasma
and proper functioning of numerous amino acid-dependent
cellular processes, including protein synthesis, energy
metabolism, and cell preservation (Christensen, 1990;
that cannot sufficiently synthesize certain amino acids and
hence require their uptake from the extracellular space for
growth and viability. Normal and malignant lymphoid cells, for
cysteine, thereduced form oftheamino acid(Eagle et al., 1966;
Iglehart et al., 1977; Ishii et al., 1981b; Gmunder et al., 1990).
Whilecysteineisrequired forgeneral proteinbiosynthesis,itis
particularly important as a rate-limiting precursor in the
biosynthesis of glutathione, a tripeptide thiol consisting of
glutamate, cysteine, and glycine, which plays a critical role in
cellular defenses against oxidative stress as a free radical
scavenger and detoxifying agent (Griffith, 1999). In cancer cells
glutathione content is particularly relevant in regulating DNA
synthesis, growth, and multidrug and radiation resistance. As
such, glutathione is considered an important target in cancer
therapy (Estrela et al., 2006). Since intracellular glutathione has
a short half-life, cysteine deficiency can readily lead to
glutathione depletion followed by growth arrest and reduced
therapy resistance. Cystine/cysteine starvation of target cells
has therefore been suggested for use in therapy of a variety of
2005; Doxsee et al., 2007).
and its role in various diseases, particularly cancer. This
transporter is essential for maintenance of a variety of
experimental cancers that require extracellular cystine/
levels in their extracellular compartment and/or (ii) their
uptake of cystine. Inhibition of the x?
be useful for generating cystine/cysteine starvation of such
cancers (Gout et al., 1997, 2001; Narang et al., 2003; Chung
et al., 2005; Doxsee et al., 2007). Furthermore, the
glutathione-based drug resistance (Okuno et al., 2003; Huang
et al., 2005; Kagami et al., 2007; Narang et al., 2007), glioma-
induced excitotoxicity (Sontheimer, 2003), and promotion of
subunit of the x?
cellular receptor for human herpesvirus 8, a causative agent of
Kaposi’s sarcoma and other lymphoproliferative syndromes
(Kaleeba and Berger, 2006). The x?
therapy of a variety of disorders.
ctransporter could hence
ctransporter appears to be critically involved in
ctransporter has been reported to act as a
ctransporter is also
Function and structure
and L-glutamate in human fibroblasts (Bannai and Kitamura,
1980). The x?
Makowske and Christensen (1982). The x?
ctransporter was first described in 1980 by Bannai and
cdesignation was subsequently assigned by
ctransporter is an
*Correspondence to: Peter W. Gout, Department of Cancer
Endocrinology, British Columbia Cancer Agency—Research
Centre, 675 West 10th Avenue, Vancouver, BC, Canada V5Z 1L3.
Received 27 August 2007; Accepted 7 November 2007
? 2 0 0 8 W I L E Y - L I S S , I N C .
obligate, electroneutral, anionic amino acid antiporter. The
exchange for intracellular glutamate with a stoichiometry of
1:1 (Fig. 1) (Bannai, 1986; Christensen, 1990; Chillaron et al.,
2001). Once inside a cell, cystine is rapidly reduced to
cysteine, which can then enter glutathione and protein
biosynthetic pathways. As a result the intracellular levels of
cystine are much lower than its extracellular levels. In
contrast, the intracellular levels of glutamate are in general
much higher than its extracellular levels, a result of glutamine
uptake via the alanine-serine-cysteine (ASC) transporter
system. This glutamate concentration gradient is thought to
provide, at least in part, the driving force of the cystine/
glutamate exchange by x?
Christensen, 1990). The x?
using either L-cystine or L-glutamate as a substrate; in both
instances, the uptake is Cl?-dependent and Naþ-independent
(Patel et al., 2004). In view of similar affinities of cystine
is potently inhibited by L-glutamate (e.g., monosodium
glutamate) and vice versa (Bannai, 1986).
cellular uptake of cystine in particular for maintenance of
from oxidative stress and xenobiotics. Furthermore, it is
instrumental in maintaining the redox balance between
extracellular cystine and cysteine. In the extracellular milieu
predominant form of the amino acid in the circulation and
particularly in culture media (Toohey, 1975); intracellularly,
cysteine is the predominant form (Bannai and Ishii, 1988;
Christensen, 1990). Whereas cysteine can be readily taken
up by mammalian cells via, for example, the ubiquitous ASC
transportsystem (Christensen, 1990), cystine transportersare
not universally expressed by cells (Gmunder et al., 1991; Gout
et al., 2001). However, somatic cells, such as fibroblasts,
activated macrophages, and dendritic cells, express the
compartment, thus closing the loop in the redox cycling of the
amino acid (Bannai and Ishii, 1988; Eck and Droge, 1989;
Christensen, 1990; Gmunder et al., 1990; Angelini et al., 2002;
Edinger and Thompson, 2002).
c(Bannai and Ishii, 1988;
ctransporter can also mediate
cantiporter has two major functions. It mediates
ctransporter and take up extracellular cystine, reduce it
Structurally, the x?
heteromeric amino acid transporters (HATs). These
transporters are composed of a heavy subunit (HSHAT) and a
light subunit (LSHAT) coupled via a disulfide bridge. The heavy
subunit is involved in trafficking of the heterodimer to the
plasma membrane, whereas the light subunit confers transport
and substrate specificity. In the case of the human x?
transporter, a heavy subunit, designated 4F2hc (also termed
CD98), is coupled to xCT, a member of the human LSHAT
family conferring specificity for cystine. The 4F2hc subunit is a
type II membrane glycoprotein commonly expressed in cells
since it acts as a subunit for various amino acid transporters
(Chillaron et al., 2001;Verrey et al., 2004).It maybenoted that
the 4F2hc subunit of x?
HSHAT, with retention of x?
Fernandez et al., 2006).
1999 by Sato and co-workers (Sato et al., 1999). They isolated
cDNA encoding the mouse x?
peritoneal macrophages treated with diethylmaleate and
lipopolysaccharide, two potent inducers of x?
Expression of mouse x?
other for a protein, designated xCT, consisting of 502 amino
acids and 12 putative transmembrane domains as indicated by
(hxCT) was identified (Sato et al., 2000; Bassi et al., 2001; Kim
et al., 2001). The full-length hxCT gene was isolated by RT-PCR
from an undifferentiated human teratocarcinoma cell line; the
protein product with 89% identity and 93% similarity to mouse
xCT (Bassi et al., 2001). To achieve expression of x?
Xenopus oocytes, expression of both hxCT and 4F2hc subunits
was required (Sato et al., 1999; Bassi et al., 2001; Kim et al.,
2001). Similarly, cystine/glutamate transport function of cloned
hxCTin human retinalcells was shown tobe dependent onco-
expression of 4F2hc (Bridges et al., 2001). Variants of hxCT
cDNA have been reported (Sato et al., 2000; Kim et al., 2001),
tissues (Kim et al., 2001). The Human Genome Mapping
Workshop (HGMW)-approved name for hxCT is Solute
of the LSHAT family have been identified (SLC7A5-11, Asc-2,
AGT-1, and arpAT) (Verrey et al., 2004; Fernandez et al., 2005).
Extensive research has been conducted on x?
topology and substrate-binding sites (Gasol et al., 2004;
Jimenez-Vidal et al., 2004). The studies focused on the xCT
subunit considered responsible for transport of cystine and
glutamate; the 4F2hc subunit, predicted to have only a single
transmembrane domain is presumably incapable of transport
activity by itself (Palacin et al., 2000; Jimenez-Vidal et al., 2004).
Glutamate transport by 4F2hc-hxCT heterodimers in Xenopus
oocytes was found to be markedly inhibited by thiol-modifying
mercurial reagents suggesting a role for cysteine residues in
cysteine mutants generated via site-directed mutagenesis, it
was found that Cys327in the 8th putative transmembrane
domain of xCT is a functionally important residue, accessible
from the aqueous extracellular compartment and structurally
(Jimenez-Vidal et al., 2004). Based onaccessibility of singlexCT
cysteines to 3-(N-maleimidyl-propionyl)biocytin, a topological
model was proposed for xCT of 12 transmembrane domains
with both the N- and C-termini located inside the cell; the
intracellular locations of the termini were confirmed by
immunofluorescence. Furthermore, a re-entrant loop with
substrate-restricted accessibility was revealed within
intracellular loops 2 and 3 (Gasol et al., 2004).
ctransporter is a member of a family of
ccan be replaced by rBAT, another
cactivity (Wang et al., 2003;
ctransporter from mouse
cactivity in Xenopus oocytes was found
electroneutral, anionic amino acid antiporter. It is a heteromeric
protein composed of two subunits coupled via a disulfide bridge, that
and substrate specificity. The anionic form of extracellular cystine is
transported into cells in exchange for intracellular glutamate with a
stoichiometry of 1:1.
Schematic diagram showing the function of the plasma
ccystine/glutamate transporter, an obligate,
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Dependence on pH
As found with cultured fibroblasts or Xenopus oocytes injected
with 4F2hc and xCT cRNA, L-cystine transport showed a
marked dependence on pH with an optimum of about 7.5. In
contrast, L-glutamate transport was almost independent of pH.
Whereas glutamate inhibited cystine uptake independently of
pH, cystine inhibited glutamate uptake in a pH-dependent
manner (Bannai and Kitamura, 1981; Bassi et al., 2001).
Monosodium glutamate is the main exchange substrate in
to that of cystine; as such it is a potent, highly specific inhibitor
of cystine uptake. Likewise, cystine competitively inhibits
glutamate uptake via the x?
1980). By specifically inhibiting the x?
monosodium glutamate can severely inhibit or completely
arrest in vitro proliferation of malignant cells that depend for
growth on x?
however, glutamate cannot be used as a therapeutic to inhibit
cellular uptake of cystine in vivo since it is neurotoxic (Choi,
1988). Other potent inhibitors of the x?
a-aminoadipate, a-aminopimelate, homocysteate (Bannai
and Kitamura, 1981), (S)-4-carboxyphenylglycine,
L-serine-O-sulphate, ibotenate, (RS)-4-bromohomoibotenate,
and quisqualate (Patel et al., 2004). In addition, x?
for human HaCaT keratinocytes (Zhu and Bowden, 2004), and
by L-lactate, as observed with cultured rat astrocytes (Koyama
et al., 2000). Certain anti-inflammatory drugs have also been
reported to have x?
1985). In a search for x?
anticancer agents, it was first observed in our laboratory
that sulfasalazine, a disease-modifying anti-rheumatic drug
(DMARD), is a potent and quite specific inhibitor of the
transporter (Gout et al., 2001).
The pharmacology and kinetic properties of system x?
have been extensively studied by Patel and co-workers
(Patelet al.,2004)usingratastrocytomacells, L-[3H]-glutamate
to determine cellular uptake of this amino acid and glutamate
efflux as a measure of substrate activity. A wide variety of
cystine, aspartate, and glutamate analogues was examined for
glutamate uptake-inhibitory activity. In addition to identifying a
number of competitive inhibitors, such uptake blockers
could be further classified as either alternative substrates
(e.g., ibotenate) or non-substrate inhibitors [e.g.,
(S)-4-carboxyphenylglycine]. Interestingly, the latter
activity, was one of the most potent competitive inhibitors,
suggesting that distinct structural features of the transporter
the cell. It is noteworthy that substrate inhibitors of the x?
transporter, while inhibiting cystine/glutamate uptake, could
potentially increase the likelihood of excitotoxic injury by way
of an x?
view of this, non-substrate x?
glutathione depletion without glutamate efflux, could be more
useful for therapeutic applications aimed at reducing growth
and/or drug resistance of malignant cells dependent on x?
ctransporter (Bannai and Kitamura,
c-mediated uptake of cystine (Gout et al., 1997);
c-inhibitory activity (Bannai and Kasuga,
cinhibitors potentially useful as novel
cexchange-mediated glutamate efflux leading to
cinhibitors, inducing intracellular
Induction of the x?
Expression of x?
be obtained in a variety of cell systems (e.g., macrophages,
retinal, endothelial cells) by numerous stimuli, including
ccoupled to increases in glutathione levels can
electrophilic agents such as diethylmaleate (Bannai, 1984a; Kim
et al., 2001; Tomi et al., 2002, 2003), oxygen (Bannai et al.,
1989), bacterial lipopolysaccharide (Sato et al., 1995), nitric
oxide (Watanabe and Bannai, 1987; Li et al., 1999; Sato
et al., 1999; Bridges et al., 2001; Dun et al., 2006), Nrf2
protein (Bridges et al., 2004). It should be noted that the
induction of glutathione synthesis can depend on the
concentration of the stimulating agent. Thus diethylmaleate at
low concentrations (0.1 mM) can increase x?
expression and glutathione levels, but at higher concentrations
(1 mM) can act as an oxidative stressor depleting glutathione
levels, as seen in human fibroblasts (Bannai, 1984a). Increased
xCT expression can also result from deprivation of cystine or
other amino acids, involving transcriptional control mediated
by amino acid response elements (Sato et al., 2004). Increased
expression of both x?
astrocytes following incubation with dibutyryl-cAMP
(Gochenauer and Robinson, 2001).
csubunits has been reported for
Regulators of x?
The response of the xCT gene to oxidative stress or
electrophiles is mediated by a cis-acting transcriptional
regulatory element in its promoter region designated
‘‘Antioxidant Response Element’’ (ARE) or ‘‘Electrophile
Response Element’’ (EpRE). Mutational analysis of the ARE/
EpRE has shown that it is critically involved in the response
to agents such as diethylmaleate, arsenite, cadmium, and
hydroquinone (Sasaki et al., 2002). The ARE/EpRE is also
essential for expression of many other antioxidant/
detoxification genes, and treatment of mammalian cells with
e.g., electrophilic agents, can trigger a coordinated expression
The inducible expression of the xCT gene and antioxidant/
of the Cap ‘‘n’’ Collar transcription factor, Nuclear factor
erythroid 2-related factor-2 (Nrf-2) (Lee and Johnson, 2004).
Thus overexpression of Nrf2 in astrocytes in vitro resulted in a
cystine transporter, as well as a coordinated upregulation of
proteins/enzymes involved in the biosynthesis of glutathione
of glutathione (glutathione-S-transferase, glutathione
reductase), and export of the tripeptide thiol (multidrug
resistance-associated Protein-1) (Shih et al., 2003). In another
defenses by upregulating levels of Nrf2 protein as well as its
targets, including the x?
et al., 2004). Under basal conditions, Nrf2 protein is
sequestered in the cytoplasm by Kelch-like ECH-associated
protein-1 (Keap1/INrf2), a negative regulator protein
associated with the actin cytoskeleton. Upon induction,
involving oxidation of cysteine residues, Nrf2 dissociates from
which leads to transcriptional activation of antioxidant/
detoxifying genes (Mann et al., 2007). ARE-mediated gene
expression can be negatively regulated by transcription factors
(Dhakshinamoorthy et al., 2005). Activation of Nrf2 has been
implicated in conferring protection against many human
(Zhang, 2006). On the other hand, targeting Nrf-2 may assist in
decreasing expression of the x?
biosynthetic genes in malignant cells thereby lowering their
ccystine/glutamate antiporter (Qiang
ctransporter and glutathione
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cT R A N S P O R T E R A S A T H E R A P E U T I C T A R G E T
Expression of the x?
cells and tissues
ctransporter in normal
mammalian cell lines. It should be noted that the expression of
the transporter by cultured cells may not reflect the native
status of the cells, since it can be induced upon culturing
(Ishii et al., 1992). In some cells/tissues, xCT mRNA has been
determined by Northern analysis or quantitative real time
RT-PCR. xCT protein has been identified via anti-xCT
antibodies of which more preparations have recently become
available (Kim et al., 2001; Burdo et al., 2006; Kaleeba and
mRNA expression has been detected in the brain (Sato et al.,
2002; Dave et al., 2004), cultured chondrocytes (Wang et al.,
2006), endothelial cell line (MBEC4) as a blood–brain barrier
(Dave et al., 2004), intestine (Bassi et al., 2001; Dave et al.,
mesenchymal C3H10T1/2 stem cells (Iemata et al., 2007); xCT
cells (Sagara et al., 1993), primary cultures of cortical neurons
(Murphy et al., 1990) and cultured pancreatic cells (Sato et al.,
1998); xCT protein was detected in the brain (Shih et al., 2006;
La Bella et al., 2007) and in the lens of the eye (Li et al., 2007);
xCT mRNA expression was observed in the lens (Lim et al.,
xCT protein distribution was shown in the kidney and
duodenum (Burdo et al., 2006). In humans, xCT mRNA
and pancreas (Bassi et al., 2001; Kim et al., 2001); xCT protein
was also observed in the brain (Burdo et al., 2006) and x?
monocytes (Eck and Droge, 1989), macrophages (Rimaniol
et al., 2001), and antigen-presenting dendritic cells (Angelini
et al., 2002). It may be noted that of a wide variety of human
expressed in the brain, pancreatic islets, and stromal and
immune cells. Of particular interest are studies with the
following normal tissues.
Brain. Brain cells, and in particular neurons, consume high
levels of oxygen for excitatory processes and therefore
generate more oxidative stress than any other cell type.
Glutathione is thought to protect the brain by detoxifying
reactive oxygen species continuously generated during
oxidative metabolism (Dringen, 2000). Astrocytes (glial cells)
are thought to protect neurons from oxidative stress by
releasing glutathione into the extracellular compartment
a process dependent on preceding uptake of cystine via the x?
et al., 2003). Subsequently, a thiol/disulfide exchange reaction
between the extracellular glutathione and cystine is thought to
cells for intracellular glutathione biosynthesis (Wang and
Cynader, 2000). Alternatively, brain cells may directly take up
cystine via the x?
primary neuronal cultures due to accumulation of cellular
oxidants (Murphy et al., 1989).
A numberof x?
to be enriched in fetal and adult rat brain at the CSF–brain
barrier (i.e., meninges) and also expressed in the cortex,
ctransporter for production of glutathione,
clocalization studies havebeen reported. Ina
hippocampus, striatum, and cerebellum (Shih et al., 2006).
Subcellular fractionation of rat brain cells showed that xCT
consistent with its structure as a transmembrane protein
(La Bella et al., 2007). In another recent study, xCT protein
in mouse and human brain was localized predominantly to
the brain proper and its periphery, including vascular
endothelial cells, ependymal cells, choroid plexus, and
leptomeninges, were also highly positive (Burdo et al., 2006).
Previously, xCT mRNA expression was found in similar
the meninges and some circumventricular organs suggesting
that the x?
fluid (Sato et al., 2002). xCT mRNA was also expressed in
mouse brain endothelial MBEC4 cells (Hosoya et al., 2002).
Together, the data suggest that the x?
contributes to the maintenance of intracellular cysteine and
glutathione levels in many areas of the brain and hence to its
In addition to its role in cystine uptake, the x?
also mediates the export of glutamate, the exchange substrate
a potent neurotoxic molecule both in vivo and in vitro (Choi,
1988; Meldrum and Garthwaite, 1990). In view of this, the
normal cellular function to occur (Burdo et al., 2006; Augustin
et al., 2007). Seizures and neuronal cell death can be induced
by an increase in the levels of extracellular glutamate. This is
normally prevented by astrocytes through glutamate uptake
via Naþ-dependent glutamate receptors such as GLT-1 and
GLAST (Ye et al., 1999). Indeed, glutamate has been implicated
as the proximate cause of many CNS pathologies, including
cerebral ischemia (stroke), seizures, hypoxia, trauma, and
hypoglycemia (Choi, 1988).
In Alzheimer’s disease, microglia can enhance the toxicity of
neurotoxic amyloid-b aggregates by releasing glutamate via
and in this context it is of interest that the x?
been implicated in the uptake of aluminum citrate by cultured
rat endothelial brain cells (Nagasawa et al., 2005). While
neuronal death may be induced by glioma cells releasing
quisqualic acid (a known glutamate receptor agonist) to
depolarization by certain excitatory amino acids and the x?
transporter has been implicated in the cellular internalization
of quisqualic acid thought to be required for the sensitization
(Chase et al., 2001). There is also evidence that the x?
transporter plays a role in virus-induced encephalopathy
(Espey et al., 1998), periventricular leukomalacia (Oka et al.,
1993), and cocaine relapse (Baker et al., 2003; Lu et al., 2004).
It is becoming increasingly evident that the x?
glutamate antiporter plays an important role in many CNS
action of this transporter could lead to development of novel
therapeutics aimed at brain diseases associated with depletion
of glutathione, including Parkinson’s disease and amyotrophic
lateral sclerosis (Schulz et al., 2000).
Pancreas. Two distinct mechanisms for L-cystine uptake
have been reported to exist in the normal human pancreatic
with the remainder being mediated via the g-glutamyl cycle by
g-glutamyl transpeptidase, an enzyme located on the outer
ctransporter may contribute to the maintenance of
ctransporter (Qin et al., 2006). Alzheimer’s disease has
JOURNAL OF CELLULAR PHYSIOLOGY
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surface of the plasma membrane (Sweiry et al., 1995).
Incubation of cultured pancreatic AR42J acinar and bTC3 islet
cells with diethylmaleate, an agent known to activate cellular
antioxidant responses, led to cystine uptake which was
predominantly mediated by the x?
defenses in pancreatic disease (Sato et al., 1998).
Ocular tissue. Oxidative damage of eye proteins is
thought to underlie major eye diseases. Glutathione may
prevent the development of such diseases by protecting thiol
groups of proteins and hence minimize oxidation-induced
protein aggregate formation via non-scheduled disulfide bond
cross-linkages. Glutathione appears to play a major role in
maintaining lens transparency and in the prevention of diseases
and cataracts (Bridges et al., 2001; Lim et al., 2005; Li et al.,
three amino acids required for biosynthesis of glutathione, are
expressed in the rat lens. Their molecular identification and
particular localization in the eye tissues are consistent with a
(Lim et al., 2005, 2006). In studies of non-infectious AIDS
retinopathy it was found that the HIV-1 genome-encoded
transactivator protein, Tat, decreased glutathione levels in the
mouse retina and upregulated the x?
enhanced release of glutamate into the extracellular space of
the retina and excitotoxicity. It is speculated that this
upregulation of the x?
pathogenesis of AIDS retinopathy (Bridges et al., 2004).
Skin. In melanocytes, cysteine is an important precursor of
to form sulfur-containing pheomelanin, the red/yellow pigment
that, together with eumelanin, determines the color of the skin
of a mouse pigmentation mutant, designated subtle gray (sut),
revealed a mutation in the SLC7A11 gene encoding the xCT
subunit of the x?
sut-mutant melanocytes showed markedly reduced uptake of
extracellular cystine and undetectable levels of glutathione.
were substantially depressed. The study suggests that the
SLC7A11 gene is a major genetic regulator of pheomelanin in
hair and melanocytes with minimal effects on eumelanin
(Chintala et al., 2005). In view of this, the x?
have yet undefined roles in melanin-related normal and
by antigen-presenting cells (activated macrophages, dendritic
cells) has been reported to involve expression of the x?
transporter in the antigen-presenting cells. This allows such
to cysteine and secrete cysteine which can be readily taken up
this process is apparently essential for lymphocyte clonal
expansion (Gmunder et al., 1990; Sido et al., 2000; Angelini
et al., 2002; Edinger and Thompson, 2002). As such, activated
macrophages and dendritic cells may serve as local suppliers
of cysteine which in general is readily taken up by cells in
1991). Similarly, stromal cells, such as fibroblasts, can
serve as cysteine suppliers by continuously secreting
cysteine. In fact, this ability was initially applied in vitro to
support growth of lymphoma cells in co-cultures with
fibroblasts (Ishii et al., 1981b). Such growth-supporting,
cysteine-secreting somatic cells could later be replaced by
2-mercaptoethanol (50–100 mM), a thiol which allows cellular
uptake of cystine via the L transport system in the form of a
mixed cysteine-2-mercaptoethanol disulfide. Once inside the
cactivity would likely contribute to cellular antioxidant
c), glycine (GLYT1), and glutamate (ASCT2), the
ctransporter. This led to
ctransporter may underlie the
ctransporter (Chintala et al., 2005). Cultured
cell, this disulfide is split into cysteine and 2-mercaptoethanol
after which the latter diffuses out of the cell. Hence 2-
mercaptoethanol provides a pathway for shuttling cystine
into cells while circumventing the x?
1981a). Use of 2-mercaptoethanol enabled establishment of a
prolactin-dependent rat Nb2 lymphoma cell line (Gout et al.,
1980) that serves as a novel, specific, and sensitive in vitro
bioassay for lactogenic hormones (Tanaka et al., 1980) and
transporter as a target for cancer therapy (Gout et al., 1997).
ctransporter (Ishii et al.,
Other cystine or glutamate transporters
Other systems mediating uptake of cystine in mammalian
cells have been reported. One, termed system b0,þ, exists as a
heterodimer consisting of either 4F2hc (SLC3A2) or rBAT
(SLC3A1) as the heavy chain and b0,þAT (SLC7A9) as the light
but includes several cationic and neutral amino acids and is
also Naþ-independent. Its expression, however, is limited
mostly to the small intestines and kidney (Rajan et al., 2000;
transport of cystine has been reported (Sweiry et al., 1995).
Additional systems mediating influx of glutamate include
the Naþ-dependent X?
excitatory amino acid transporters (EAAT1-5) of glutamate
and aspartate (Kanai and Hediger, 2004). The X?
transporters can also mediate cellular uptake of cystine
(McBean and Flynn, 2001). The x?
has been found to be highly dominant in a variety of systems,
macrophages (Gmunder et al., 1990), dendritic cells (Angelini
et al., 2002), and fibroblasts (Bannai and Ishii, 1988). It may be
noted that although the x?
health, especially in vitro, it is dispensable during mammalian
development, as demonstrated with the generation of healthy
and fertile x?
AGfamily, commonly known as the
ctransporter can be vital to cellular
cknock-out mice (Sato et al., 2005).
Role of the x?
Growth requirement of cancers for extracellular cystine/
cysteine: Leukemias and lymphomas
cCystine/Glutamate Antiporter in Cancer
Cysteine is traditionally viewed as a nutritionally non-essential
amino acid since it is synthesized in the body, primarily by
the liver, from L-methionine via the transsulfuration pathway
(Rosado et al., 2007). Certain cancers, including leukemias and
lymphomas, are incapable of synthesizing cysteine (Iglehart
et al., 1977; Gout et al., 1997), probably due to a deficiency
in g-cystathionase, the last enzyme in the transsulfuration
pathway (Uren and Lazarus, 1979). Cystine/cysteine is thus an
essential amino acid for such cancers and its uptake from the
micro-environment is vital for their growth and viability.
A growth requirement for extracellular cystine can be readily
medium specifically deficient in the amino acid and monitoring
enhancers (e.g., 2-mercaptoethanol) from the medium
(Gout et al., 1997). Blood plasma contains relatively high
concentrations of cystine (100–200 mM half-cystine), but
only 10–20 mM cysteine; such cysteine concentrations are
extremely low in comparison with the plasma concentrations
of other protein-forming amino acids (Saetre and Rabenstein,
generally have a low uptake capability for cystine due to lack
of cystine transporter expression and, in the absence of
endogenous cysteine-synthetic ability, are mainly dependent
on uptake of extracellular cysteine (Gmunder et al., 1991;
Gout et al., 1997). In view of the relatively very low cysteine
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cT R A N S P O R T E R A S A T H E R A P E U T I C T A R G E T
concentrations in the circulation it is thought that—in vivo—
such cells acquire the necessary amounts of cysteine for their
functions from transient increases in the levels of this amino
in their vicinity (e.g., fibroblasts, activated macrophages, or
dendritic cells)—a process based on x?
uptake (Gmunder et al., 1990; Sido et al., 2000; Angelini
et al., 2002; Edinger and Thompson, 2002). Furthermore,
tumor-associated macrophages(TAMs) have beenreported to
promote cancer growth via secretion of a variety of factors,
including cytokines and angiogenic factors (Bingle et al., 2002).
resistance of certain cancers via secretion of cysteine essential
cysteine by somatic cells and in the uptake of cystine by target
cells led to an early suggestion that the x?
represents a potential target for therapy of cancers that are
viability (Gout, 1997; Gout et al., 1997).
Expression of x?
cultured cancer cell lines, including human/rat hepatoma cells
(Makowske and Christensen, 1982; Bannai, 1984b; Maechler
and Wollheim, 1999), advanced rat and human lymphoma cells
(Gout et al., 2001), human glioma cells (Chung et al., 2005) and
human colon (Bassi et al., 2001), breast (Narang et al., 2003),
prostate (Doxsee et al., 2007), and pancreatic (Lo et al., 2006)
cancer cells. In some cases, the presence of the x?
in cultured cell lines reflects its regular expression in their
tissues of origin, such as normal pancreatic tissue (Bassi et al.,
2001). However, the presence of the transporter in cultured
cells, whenthetissues oforigindonot normallyexpress it, may
be a manifestation of cells circumventing cystine starvation.
Whereas, in vivo, cells have access not only to cystine but also
to cysteine, culture media such as Minimum Essential Medium
and Fischer’s medium only provide cystine [cysteine would be
rapidly converted to cystine via autoxidation (Toohey, 1975)].
Establishment in such media of cell lines from cancer tissue,
requiring extracellular cystine/cysteine but lacking cystine
transporters, may therefore be due to cellular adaptation
leading to x?
subpopulation of x?
lymphoma cells (Hishinuma et al., 1986; Gout, 1987) and a
neutrophils did not express x?
established in vitro (Sakakura et al., 2007). It may be noted that
markedly exceeding those in for example, Minimal Essential
boost their culture growth sustaining ability. Cellular uptake
of cystine can also be enhanced by additives such as
when they have been established in medium containing
2-mercaptoethanol; advanced lymphoma cells expressing
the transporter have no requirement for cystine uptake
Gout et al., 1997, 2001).
The existence of x?
recently been established. Thus, as shown by Western blot
analysis, both 4F2hc and xCT subunits were prominently
expressed in all glioma samples acutely derived from five
patients (Lyons et al., 2007).
cin cancer cell lines and tissues
ctransporter has been demonstrated in numerous
cexpression or to outgrowth of an existing
c-expressing cells, as reported for
cactivity until the cells were
c-expressing cancers in patients has
in normal brain (Bassi et al., 2001; Kim et al., 2001; Burdo et al.,
2006) raises the possibility that it plays a role in the growth and
patient-derived non-malignant brain tissues and glioma tissues
(Ye and Sontheimer, 1999; Ye et al., 1999; Chung et al., 2005;
Lyons et al., 2007). In glioma cells the transporter was found to
represent the only viable pathway for cystine uptake to sustain
glutathione synthesis and growth. This dependence on cystine
uptake was exploited in inhibiting experimental glioma growth
in vitro and in vivo (Chung et al., 2005). Gliomas are aggressive
cancers for which no effective treatment exists. Although their
they are able to overcome this physical limitation by killing
neurons in their vicinity, thus vacating space; gliomas are
therefore often associated with seizures (Sontheimer, 2003;
Chung et al., 2005). The destruction of neuronal tissue
apparently occurs, at least in part, by release of excessive
amounts of glutamate (Ye and Sontheimer, 1999; Takano et al.,
transporter activity, since over 50% of glutamate release by
(Ye et al., 1999). The glutamate release in vivo is an obligatory
byproduct in the x?
glutamate for extracellular cystine, as the latter is required by
the glioma cells for glutathione-based protection against
reactive oxygen species (Chung et al., 2005). However, the
elevated extracellular glutamate concentrations are not only
due to enhanced x?
their reduced glutamate uptake capability relative to normal
glial cells (Murphy et al., 1990; Ye et al., 1999; Schubert and
Piasecki, 2001; Sontheimer, 2003; Lewerenz et al., 2006;
Domercq et al., 2007). Recent evidence has indicated that
glutamate, released by malignant glioma cells via the x?
transporter, can also act as an autocrine/paracrine signal
promoting glioma cell invasion and that this process can be
inhibited with potent blockers of the x?
sulfasalazine and (S)-4-carboxyphenylglycine (Lyons et al.,
ctransporter have been reported in glioma cell lines,
c-mediated exchange of intracellular
ctransport activity of gliomas, but also to
csystem such as
Recent studies in our laboratory have shown that the x?
transporter is markedly expressed in a number of human
pancreatic cancer cell lines (Lo et al., 2006), a finding not
unexpected since x?
pancreatic tissue (Bassi et al., 2001; Kim et al., 2001). The
growth of these cell lines is critically dependent on uptake of
extracellular cystine and can be arrested by cystine starvation
(Lo et al., 2006).
cis substantially expressed in normal
Recently, the xCT subunit of the x?
play a role in Kaposi’s sarcoma, a connective tissue cancer
be the predominant receptor in host cells mediating the fusion
and entry of Kaposi’s Sarcoma-associated Herpesvirus (KSHV)
8 (Kaleeba and Berger, 2006). Interestingly, inhibition of the
transporter, resulting from its interaction with the virus, could
lead to depletion of intracellular glutathione which, in turn,
would be expected to result in upregulation of the transporter
receptors for viral entry of the host and enhancing KSHV
infectivity (Kaleeba and Berger, 2006). This discovery of an
could lead to novel x?
ctransporter was found to
c-targeted therapy of the disease.
JOURNAL OF CELLULAR PHYSIOLOGY
L O E T A L .
Early evidence for a role for the x?
progression came from a rat prolactin-dependent Nb2
model for the malignant progression of lymphomas to growth
autonomy (Gout et al., 1994). The sublines had clonally
developed from the parent line with an increase in the number
et al., 1991). The emergence of x?
the Nb2-SFJCD1 subline and contrasting with the lack of x?
activity in the parent line, augmented the cystine uptake and
glutathione-generating capability of this subline thereby
enhancing its growth autonomy (Gout et al., 1997) and
resistance against oxidative stress (Meyer et al., 1998),
typical features of malignant progression.
ctransporter in tumor progression
ctransporter in tumor
cexpression, manifested in
Role of x?
mediating cellular uptake of cystine to enhance biosynthesis of
glutathione, as found for ovarian (Okuno et al., 2003) and lung
(Huang et al., 2005) cancer cells. This tripeptide thiol has a
major role in the protection of cells from drug-induced
oxidative stress by mediating cellular detoxification of drugs
and their extrusion via multidrug resistance proteins (Haimeur
et al., 2002; Filipits et al., 2005; Yang et al., 2006; Yadav et al.,
2007). Apparently, glutathione induces a conformational
change within the multidrug resistance-associated protein-1
(MRP1) which is essential for interaction of this efflux
protein with a drug and extrusion of the latter (Uchino
et al., 2002). Conversely, depletion of intracellular glutathione
levels, as induced by treatment with for example,
L-buthionine-(S,R)-sulfoximine (BSO), an inhibitor of
glutathione biosynthesis, can cause marked inhibition of cell
growth, induction of apoptosis (Schnelldorfer et al., 2000), and
would provide an alternative avenue for overcoming drug
resistance, as previously suggested (Gout et al., 2003).
In a pharmacogenomics approach, microarrays have been
used to analyze gene expressions of membrane transporters in
60 human cancer cell lines, used by the National Cancer
Institute for drug screening (NCI-60), and the expression of
revealed 39 drugs showing positive expression correlations
with the x?
(e.g., geldanamycin). These findings highlight the possibility of
using L-alanosine-related drugs as a potential therapy for
cancers expressing the x?
be expected with drugs structurally similar to geldanamycin
(Huang and Sadee, 2006). Importantly, such pharmacogenomic
studies may be used to predict mechanisms of drug sensitivity
and resistance and provide insights for selecting optimal drug
regimens for combination chemotherapy (Dai et al., 2007).
cin multidrug resistance
ctransporter was linked with the potencies of 1,400
ctransporter (e.g., the amino acid analogue,
ctransporter, while resistance may
Cystine/cysteine starvation of target cells via inhibition
of the x?
ccystine transporter: Use of sulfasalazine
of major importance in cancer management and many different
approaches have been initiated, including nutrient starvation of
targetcells. Thusasparaginasetreatment, aimedatdepletion of
L-asparagine in the circulation, has been used for decades in
clinical combination chemotherapy of acute lymphocytic
leukemia cells critically depend on extracellular cystine/
cysteine for growth (Iglehart et al., 1977) suggested that
depletion of this amino acid in the circulation provided a
useful therapeutic approach for such malignancies. In fact,
a therapeutic regimen was developed based on an
cysteine biosynthesis. Clinical trials were precluded, however,
by rapid plasma clearance of the enzyme (Uren and Lazarus,
1979). In exploring an alternative method to attain cystine/
cysteine starvation of lymphomas, it was recognized by one of
us (PWG) that this could probably be achieved by specifically
cysteine supply by somatic cells as well as uptake of cystine by
target cells (Gout, 1997), as illustrated in Figure 2. Support for
this approach was obtained in vitro. Monosodium glutamate
and Kitamura, 1980), markedly reduced cystine uptake by
Nb2-SFJCD1 lymphoma cells and almost completely arrested
(98%) their proliferation in Fischer’s medium. This growth
arrest was based on specific inhibition of cystine uptake since it
could be essentially completely prevented by inclusion in the
medium of 50 mM 2-mercaptoethanol aimed at supplying the
(Gout et al., 1997). Unfortunately, monosodium glutamate is a
neurotoxin which precludes its use as a therapeutic agent
(Choi, 1988). In a search for a compound that inhibited the
transporter and could be used therapeutically, it was
discovered in our laboratory that the anti-inflammatory drug,
sulfasalazine, potently inhibited x?
by Nb2-SFJCD1 lymphoma cells (Gout et al., 2001). In vitro,
0.1–0.3 mM sulfasalazine, a concentration range found in
patients’ sera, markedly arrested the proliferation of rat
Nb2-SFJCD1 and human DoHH2 lymphoma cells whose
growth in vitro depends on proper x?
c-mediated uptake of cystine
cfunctioning. The growth
required for growth and/or therapy resistance. In the extracellular
milieu cysteine is quickly oxidized to cystine, leading to cystine
predominance andlow cysteine levels. Cysteine isreadily taken up by
cells, due to the expression of ubiquitous cysteine transporters; in
contrast, cystine is not, since cystine transporters are not expressed
by all cell types. Tumor-associated somatic cells, such as activated
macrophages and dendritic cells (involved in the immune response),
can regenerate cysteine from cystine, by taking up cystine via
micro-environment. By using a specific x?
uptake of cystine by the somatic cells can be inhibited, leading to
reduced levels of cysteine in the environment of the cancer cells. The
inhibitor will also reduce cystine uptake by cancer cells when they
express the x?
the cancer cells would readily lead to intracellular GSH depletion,
subsequent growth arrest, and/or reduced drug resistance.
ctransporter as a target for induction of cystine/
ctransporter and secreting it as cysteine back into the
cinhibitor (INHIB), the
ctransporter. INHIB-induced cysteine deficiency of
JOURNAL OF CELLULAR PHYSIOLOGY
cT R A N S P O R T E R A S A T H E R A P E U T I C T A R G E T
arrest and subsequent cell lysis were primarily due to cystine
starvation since they could be largely prevented by 2-
mercaptoethanol (60 mM). Sulfasalazine (0.15 mM) also
inhibited secretion by fibroblast feeder layers of cysteine,
critical for the growth of co-cultured Nb2 lymphoma cells
lacking the x?
not inhibited. Importantly, intraperitoneal administration of
substantial growth arrest of well-developed, rapidly growing
hosts; it may be noted that the Nb2-U17 cells do not express
found that sulfasalazine induced marked glutathione depletion
(>90%) in human DU-145 and PC-3 prostate and Mia PaCa-2
and Panc-1 pancreatic cancer cells leading to growth arrest;
growth of xenografts of these cell lines in SCID mice (Lo et al.,
2006; Doxsee et al., 2007). Furthermore, sulfasalazine
significantly enhanced the growth-inhibitory activity of
doxorubicin against human MDA-MB-231 mammary cancer
cells in vitro (Narang et al., 2007) and PC-3 prostate cancer
xenografts (Kagami et al., 2007). Taken together, the above
viable strategy for chemotherapy of a variety of cancers,
acute lymphocytic leukemia may, in view of its lymphoid origin,
respond to cystine/cysteine starvation and its treatment might
be improved by using asparaginase in combination with an
Sulfasalazine has also been used as an anticancer agent by
other researchers. Quite recently Robe et al. (2004) showed,
using xenograft animal models for human gliomas, that
sulfasalazine could significantly inhibit glioma growth in vivo.
A tumor-sensitizing effect of sulfasalazine was reported for
human pancreatic cancer xenografts by Muerkoster et al.
by Lay et al. (2007). The anticancer effects of sulfasalazine in
these studies were thought to be based on its inhibitory effect
on activation of the transcription factor, NFkB (Wahl et al.,
1998), considered an important target for cancer growth and
chemoresistance (Arlt et al., 2003). Other researchers have
thought to act via inhibition of glutathione S-transferase, a
detoxification enzyme (Awasthi et al., 1994), and a limited
clinical trial, using sulfasalazine in combination with melphalan,
showed some success for treatment of ovarian cancer (Gupta
et al., 1995).
In contrast to the studies by Robe et al. (2004), more recent
growth could be inhibited by treatment with sulfasalazine, both
in vitro and in a xenograft model, but rather as a consequence
(Chung et al., 2005). These findings are in line with our
observations that the growth-inhibitory effects of sulfasalazine
(0.05–0.4 mM) in vitro are primarily due to glutathione
depletion resulting from cystine/cysteine starvation induced by
inhibition of the x?
growth arrest could be prevented by specifically enhancing
cellular uptake of cystine via a route circumventing the x?
transporter (Gout et al., 2001; Narang et al., 2003; Lo et al.,
2006; Doxsee et al., 2007). While sulfasalazine-induced cell
et al., 2005), recent evidence in our laboratory indicates that
sulfasalazine-induced death of human PC-3 prostatic cancer
cells can involve autophagy (Kagami et al., unpublished work).
ctransporter; this led to growth arrest of the
ctransporter (Gout et al., 2001, 2003). Subsequently we
cinhibitor such as sulfasalazine.
cinhibition leading to reduced glutathione levels
ctransporter. In fact, sulfasalazine-induced
glioblastomas and a SPORE clinical trial has been initiated by
Sontheimer et al. (2006) on potential use of sulfasalazine for
preventing neurotoxic glutamate release by gliomas.
cellular cystine uptake, an important role in maintaining
health of both normal and malignant cells. With regard to the
latter there is an increasing body of evidence that short-term,
specific inhibition of the x?
growth and/or reduced drug resistance of a variety of cancers
without major side effects to the host. Accordingly, relatively
conventional chemotherapeutics. The mediation of glutamate
efflux by the antiporter is apparently a contributing factor in
excitotoxicity associated with a variety of brain disorders,
including Alzheimer’s disease and malignant gliomas, and
useful in this area. Of recent interest is the finding that the
therapeutic target for a number of diverse diseases.
ccystine/glutamate antiporter has, as a mediator of
ctransporter can result in inhibited
c-inhibitory drugs such as sulfasalazine may prove
cinhibitors may therefore also be therapeutically
ctransporter can act as a cellular receptor mediating the
ctransporter therefore appears to provide a potential
The BC Cancer Foundation (Vancouver, BC) is thanked for
their long-term, donation-based support of PWG.
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