Mol. Cells, Vol. 14, No. 2, pp. 305-311
Communication
M olecules
and
Cells
KSMCB 2002
Activities of Antioxidant and Redox Enzymes in Human Normal
Hepatic and Hepatoma Cell Lines
Yuk-Young Lee, Hong-Gyum Kim, Haeng-Im Jung, Youn Hee Shin, Sung Min Hong, Eun-Hee Park1,
Jae-Hoon Sa2, and Chang-Jin Lim*
Division of Life Sciences, Kangwon National University, Chunchon 200-701, Korea;
1
College of Pharmacy, Sookmyung Women’s University, Seoul 140-742, Korea;
2
Department of Food and Drug Analysis, Kangwon Institute of Health and Environment, Chunchon 200-093, Korea.
(Received May 10, 2002; Accepted July 19, 2002)
The cellular defense system (including glutathione,
glutathione-related enzymes, antioxidant and redox
enzymes) plays a crucial role in cell survival and
growth in aerobic organisms. To understand its
physiological role in tumor cells, the glutathione content and related enzyme activities in the human normal hepatic cell line, Chang and human hepatoma cell
line, HepG2, were systematically measured and compared. Superoxide dismutase, catalase, and glutathione
peroxidase activities are 2.8-, 4.3-, and 2.9-fold higher
in HepG2 cells than in Chang cells. Total glutathione
content is also about 1.4-fold higher in HepG2, which
LV VXSSRUWHG E\ VLJQLILFDQW LQFUHDVHV LQ -glutamylcysteine synthetase and glutathione synthetase activities.
Two other glutathione-related enzymes, glutathione
UHGXFWDVH DQG -glutamyltranspeptidase, are upregulated in HepG2 cells. However, thioredoxin reductase and glutathione S-transferase activities are significantly lower in HepG2 cells. These results propose
that defense-related enzymes are largely modulated in
tumor cells, which might be linked to their growth and
maintenance.
Keywords: Catalase; Chang; Glutathione; Glutathione
Peroxidase; HepG2; Superoxide Dismutase; Thioredoxin
Reductase.
Introduction
Demo
Oxidative stress is an unavoidable consequence of oxygen
metabolism, and occurs in aerobic cells. The reactive oxy* To whom correspondence should be addressed.
Tel: 82-33-250-8514; Fax: 82-33-242-0459
E-mail: [email protected]
gen species (ROS), such as superoxide anions (O2−), hydrogen peroxide (H2O2), and hydroxy radicals (OH⋅), are
produced by both normal aerobic metabolism and environmental agents. They can damage intracellular components, such as DNA, proteins, and membrane lipids (Ross
et al., 2000; Smirnova et al., 2000). Consequently, this
can result in mutagenesis, inhibition of growth and cell
death, as well as aging implications, all stages of cancer,
and numerous diseases (Halliwell, 1987; Smirnova et al.,
2000). Levels of antioxidant enzymes, such as superoxide
dismutase, catalase and peroxidase, are closely linked
with cellular responses to various oxidative stresses. The
response of cells to oxidative stress depends on the severity of the stimulus. Massive oxidative stress is uniformly
detrimental. While antioxidant enzymes are highly efficient, oxidative damage still occurs in cells under normal
physiological conditions. The transgenic overexpression
of superoxide dismutase and catalase have a marked protected effect, such as the prolongation of both average and
maximal life span in Drosophila (Orr and Sohal, 1994;
Sohal et al., 1995).
Reduced glutathione (GSH), the major intracellular
thiol, is believed to be an important protector against free
radical damages by providing reducing equivalents for
antioxidant enzymes and also by scavenging hydroxyl
radicals and singlet oxygen. The GSH level increases in
hepatocytes during active proliferation; increased GSH is
due to the enhanced expression of GSH synthetic enzymes (Huang et al., 2001). Several studies reported that
chemicals that generate oxidative stress can result in inFUHDVHG JOXWDWKLRQH OHYHOV DQG -glutamylcysteine synthetase activity (Galloway et al., 1999). The plasma
membrane-ERXQGHQ]\PH-glutamyltranspeptidase initiAbbreviations: GST, glutathione S-transgerase; ROS, reactive
oxygen species; TRX, thioredoxin.
306
Antioxidant and Redox Enzymes in Human Cell Lines
DWHV WKH EUHDNGRZQ RI *6+ E\ UHPRYLQJ WKH -glutamyl
moiety, and therefore providing amino acid precursors for
the intracellular de novo synthesis of GSH (Enoiu et al.,
2000)7KH DFWLYLW\RI-glutamyltranspeptidase increases
in a regenerating rat liver, but the increase is out of phase
with the proliferative response (Sulakhe, 1986).
Glutathione S-transferase (GST) catalyzes the addition
of glutathione moiety to a great variety of acceptor molecules. The acceptors include carcinogens, organic hydroperoxides, and lipid peroxides, which cause oxidative
stress. The conjugation is usually considered a detoxificaWLRQ WKH FRQMXJDWH LV GHJUDGHG E\ WKH HQ]\PH RI WKH glutamyl cycle. In addition to alleviating the cytotoxic
effects of oxidative stress, GST may interfere with the
subtoxic but cytostatic signals that are generated by a
low-level pro-oxidant state (Zimniak et al., 1997).
Redox regulation plays an important role at diverse levels in cellular functions, including stress response and cell
growth. Thioredoxin (TRX) and thioltransferase (TTase)
are small disulfide reducing enzymes, which have conserved consensus sequences -CXXC- at their active site.
TRX is a potent protein disulfide oxidoreductase, which is
important in antioxidant defense, the regulation of cellular
proliferation, and the regulation of gene expression
through transcription factor activation (Hayashi et al.,
1993; Nakamura et al., 1997; Powis et al., 1994). In addition, it is a powerful singlet oxygen quencher and hydroxyl radical scavenger (Das and Das, 2000). Because of
its role in stimulating cancer cell growth and as an inhibitor of apoptosis, TRX offers a target for the development
of drugs to treat and prevent cancer (Freemerman and
Powis, 2000; Powis et al., 2000). Cytosolic thioredoxin
reductase, a selenoprotein, catalyzes the NADPHdependent reduction of TRX disulfide and of numerous
other oxidized cell constituents (Becker et al., 2000). As a
general reducing enzyme with little substrate specificity,
it also contributes to redox homeostasis. It is also involved in the prevention, intervention, and repair of damage that is caused by H2O2-based oxidative stress (Becker
et al., 2000).
This investigation was carried out with the assumption
that stress response-related enzymes might be closely
linked with the growth of cancer cells. Therefore, the present studies were undertaken to evaluate and compare
various antioxidant and redox proteins in human normal
hepatic and hepatoma cell lines.
Materials and Methods
Chemicals Reduced glutathione (GSH), oxidized glutathione
(GSSG), 1-chloro-2,4-dinitrobenzene (CDNB), 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB), glutathione reductase (GR),
phosphoenolpyruvate, L-glutamate, L-α-aminobutyrate, pyruvate kinase, lactate dehydrogenase, Nonidet P-40, phenyl-
methylsulfonyl fluoride (PMSF), and NADPH were all purchased from the Sigma Chemical Co. (USA). Gibco-BRL
(Gaithersburg, USA) supplied Dulbecco’s modified Eagle’s
medium and the pre-mixed penicillin (10,000 units/ml) and
VWUHSWRP\FLQ JPO )HWDO ERYLQH VHUXP )%6 ZDV
obtained from Biowhittaker, Inc. (Walkersville, USA). L-Glutamyl-p-nitroanilide was from BioVectra dcl (Charlottetown,
USA). L--Glutamyl-L-α-aminobutyrate was synthesized by
Anaspec Inc. (USA). CO2 was purchased from local vectors. All
of the other chemicals that were used in the study were of analytical grade.
Cell lines The human normal liver cell line, Chang (American
Type Culture Collection CCL-13), and the human hepatocyte
carcinoma cell line, HepG2, were kindly provided by Dr. K. W.
Kim (Seoul National University, Korea). These cell lines were
grown in Dulbecco’s modified Eagle’s medium with 10% (v/v)
heat-inactivated FBS, 100 units/ml penicillin G, DQGJPO
streptomycin. All of the cells were grown at 37°C in a humidified air/CO2 (19:1) atmosphere, and harvested at a logarithmic
growth.
Preparation of cytosolic extracts Cytosolic extracts from the
human cell lines were prepared by sonication and centrifugation
at 12,000 × g for 15 min.)RUWKH-glutamyl transpeptidase activity measurement, the cultured cells were lysed by an animal
cell lysis buffer (50 mM Tris buffer, 150 mM NaCl, 0.02%
NaN3, 1% Nonidet P-40, 1 mM PMSF). Protein concentrations
were determined according to the method of Bradford (Bradford,
1976), using bovine serum albumin as a standard.
Measurement of total GSH Total GSH content was determined by the absorbance at 412 nm (Sies and Akerboom, 1984)
using GSSG as a standard. In a final volume of 0.5 ml, the reaction mixture contained a 100 mM phosphate buffer (pH 7.0), 1
mM EDTA, 0.24 mM NADPH, 0.0756 mM DTNB, and 0.06
XQLWV*57KHQORIWKHDSSURSULDWH*66*VWDQGDUGRU
µl of the crude extract, was added to each of the cuvettes. The
absorbances, obtained from known concentrations of GSSG,
were used to construct a standard curve.
Enzymatic assays GST activity was spectrophotometrically
determined as previously described (Habig et al., 1974) with
minor modifications. The reaction mixture contained a 100 mM
phosphate buffer (pH 6.5), 5.0 mM GSH, 2.0 mM CDNB, and a
crude extract in a volume of 1.0 ml. The reaction, conducted at
25°C, was initiated by the addition of CDNB. The change in the
absorbance at 340 nm was monitored with a spectrophotometer.
All of the initial rates were corrected for the background of
nonenzymatic reaction.
7KH-Glutamylcysteine synthetase activity was determined as
previously described (Seelig and Meister, 1985). Enzyme activity was determined at 37°C in reaction mixtures (final volume,
1.0 ml) that contained 0.1 M Tris-HCl buffer, 150 mM KCl, 5
mM ATP, 2 mM phosphoenolpyruvate, 10 mM L-glutamate, 10
Yuk-Young Lee et al.
mM L-α-aminobutyrate, 20 mM MgCl2, 2 mM EDTA, 0.2 mM
NADH, 17 µg of pyruvate kinase, and 17 µg of lactate dehydrogenase. The reaction waV LQLWLDWHG E\ WKH DGGLWLRQ RI glutamylcysteine synthetase. The absorbance at 340 nm was
monitored.
Glutathione synthetase activity was determined by measuring
the formation of ADP in reaction mixtures that contained the
enzyme and its substrates (Meister, 1985). The reaction mixture
contained 100 mM Tris-HCl buffer (pH 8.2 at 37°C), 50 mM
potassium chloride, 5 mM L--glutamyl-L-α-aminobutyrate, 10
mM ATP, 5 mM glycine, 20 mM magnesium chloride, 2 mM
EDTA, and extract in a final volume of 0.1 ml. The assay mixture was incubated for 2.5−30 min at 37°C. To determine ADP,
the reaction mixtures were treated with 0.02 ml of 10% sulfosalicyclic acid and 0.9 ml of a solution that contained 0.5 mM
phosphoenolpyruvate, 0.2 mM NADH, pyruvate kinase (1 unit),
40 mM magnesium chloride, 50 mM potassium chloride, and
250 mM potassium phosphate buffer (pH 7.0). The amount of
ADP that was formed was calculated from the change in absorbance at 340 nm that was observed after the addition of 0.1 ml (1
unit) of lactate dehydrogenase.
GR activity was spectrophotometrically assayed after NADPH
oxidation at 340 nm (Carlberg and Mannervik, 1985). The reaction mixture (0.2 ml) contained a 0.1 M phosphate buffer (pH
7.0), 1 mM GSSG, and 0.1 mM NADPH. The reaction was initiated by the addition of the enzyme.
7KH -Glutamyl transpeptidase activity was spectrophotometrically measured as previously described (Meister et al., 1981).
7KH UHDFWLRQ PL[WXUH PO FRQWDLQHG P0 -glutamyl-pnitroanilide, 20 mM glycylglycine, and 0.06 M Tris-HCl (pH
8.0). The reaction was initiated by adding a suitable amount of
enzyme. The rate of p-nitroaniline was recorded at 410 nm.
Catalase activity was determined by monitoring the decrease
in absorbance at 240 nm that was due to H2O2 consumption
(Cho et al., 2000; Rao et al., 1996). The 3 ml reaction mixture
contained a 100 mM phosphate buffer (pH 7.0) and 10 mM
H2O2. The mixture was incubated for 2.5 min at 30°C. The reaction was initiated by adding the crude extract.
Since thioltransferase contained transhydrogenase activity, its
activity was measured spectrophotometrically at 340 nm by the
use of GR as a coupling enzyme (Cho et al., 1998; Holmgren,
1979; Kim et al., 1998; 1999). In a total volume of 200 µl, the
reaction mixture contained 1 mg/ml of BSA, 10 mM GSH, 60
µg/µl yeast GR, 4 mM NADPH, and 1 M Tris-HCl/20 mM
EDTA, pH 8.0. The change in the absorbance was then recorded
with time.
Thioredoxin reductase activity was measured as the reduction
of DTNB in the presence of NADPH (Tamura and Stadtman,
1996). The assay mixture contained a 0.2 M phosphate buffer
(pH 7.6), 1 mM EDTA, 0.25 mM NADPH, and 1 mM DTNB.
The increase of absorbance at 412 nm was monitored over 5 min
at 25°C.
Glutathione peroxidase activity was determined as previously
described (Flohé and Günzler, 1984). The reaction mixture contained a 0.05 M phosphate buffer (pH 7.0), 0.24 units glu-
307
tathione reductase, 1 mM GSH, and an enzyme sample. The
mixture was preincubated for 10 min at 37°C. Thereafter, a 100
O 1$'3+ P0 VROXWLRQ ZDV DGGHG DQG WKH K\GURSHURxide-independent consumption of NADPH was monitored for
about 3 min. The overall reaction was started by adding 100 µl
of a prewarmed hydroperoxide solution (1.5 mM). The decrease
in absorption at 340 nm was monitored.
Total superoxide dismutase activity was spectrophotometrically determined as previously described (Punchard and Kelly,
1996). The reaction mixture (0.2 ml) contained 10−2 units/ml
xanthine oxidase, a 50 mM phosphate buffer (pH 7.4), 0.1 mM
EDTA, 1 µM catalase, 0.05 mM xanthine, 20 µM cytochrome c,
and an enzyme sample. The change in absorption at 550 nm was
monitored.
Determination of nitrite content Fresh cytosolic extract was
used to determine the nitrite content as previously described
(Hevel and Marletta, 1994). The extract (100 µl) was mixed
with 100 µl of a Griess reagent (1% sulfanilamide, 0.1%
naphthyl ethylene diamine dihydrochloride in 5% phosphoric
acid). The absorbance at 543 nm was recorded against a blank.
Statistical analysis The data was analyzed for mean values and
standard deviations. These were subjected to a statistical comparison using the Student’s t-test; p < 0.05 was considered to be
significant.
Results and Discussion
When aerobic cells grow, reactive oxygen species, and
other harmful compounds are produced from their normal
metabolic processes, and may inhibit cell growth. Defense
enzyme systems, such as antioxidant and redox enzymes,
would be required for normal cell growth. The cancerous
growth of mammalian cells could also be affected. Therefore, defense enzyme levels may play an important role in
the survival and growth of cancer cells. In this investigation, the human normal hepatic cell line, the Chang and
human hepatoma cell lines, HepG2, were chosen as models of mammalian normal and tumor cells.
Aerobic organisms possess antioxidant defense systems.
These include three well-known enzymes, superoxide
dismutase, catalase, and glutathione peroxidase. They
have a dramatic effect on the resistance of mammalian
cells to oxidative damage to lipids, proteins, and DNA.
The total superoxide dismutase activity was 0.26 ± 0.14
and 0.75 ± 0.21 in the Chang and HepG2 cell lines, respectively (Table 1). Superoxide dismutase activity is 2.8fold higher in HepG2 cells than in Chang cells. Superoxide dismutase catalyzes the dismutation of the superoxide
radical to molecular oxygen and hydrogen peroxide,
which in turn is metabolized to harmless water and oxygen by catalase and glutathione peroxidase. Catalase and
glutathione peroxidase activities were also determined in
308
Antioxidant and Redox Enzymes in Human Cell Lines
Table 1. Antioxidant enzyme activities in Chang and HepG2
cell lines.
Table 2. Glutathione, glutathione-synthesizing and glutathionemetabolizing enzymes in Chang and HepG2 cell lines.
Specific activitiesa
Specific activitiesa
Enzymes
Enzymes
Chang
Superoxide dismutaseb
Catalasec
Glutathione peroxidased
HepG2
0.26 ± 0.14 (100) 0.75 ± 0.21 (280)*
0.10 ± 0.02 (100) 0.44 ± 0.13 (430)*
1.05 ± 0.58 (100) 3.07 ± 0.24 (290)*
a
Values represent mean±SD. Numbers in parentheses indicate relative specific activities.
b
Superoxide dismutase activity was represented as ∆A412/min/mg
protein.
c
Catalase activity was represented as ∆A240/min/mg protein.
d
Glutathione peroxidase activity was represented as ∆A340/min/mg
protein.
*, p < 0.001.
two cultured cell lines, according to spectrophotometric
methods. Catalase activity was 0.10 ± 0.02 and 0.44 ±
0.13 in Chang and HepG2 cell lines, respectively (Table
1). It was 4.3-fold higher in the hepatoma cell lines than
in the normal hepatic cell lines. Glutathione peroxidase
activity was detected to be 1.05 ± 0.58 and 3.07 ± 0.24 in
Chang and HepG2 cells, respectively. It appeared to be
2.9-fold higher in HepG2 cells than in the normal hepatic
cells. Collectively, specific activities of all three antioxidant enzymes were identified to be at least 2.8-fold higher
in the HepG2 cells than in the Chang cells. The mechanism of the increased antioxidant enzyme levels remains
to be elucidated. Their genes might be under coordinate
control circuit.
A high concentration of GSH is present in most living
cells from microorganisms to humans. It is known to be
involved in the responses to various stresses (Pennincks,
2000). GSH also plays a vital role in defending against
toxins, as well as storing and transferring cysteine (DeLeve and Kaplowitz, 1991). Its synthesis occurs via two
enzymes of-glutamylcysteine synthetase and glutathione
synthetase. The total GSH levels of the Chang and HepG2
cell lines were determined to be 22.74 ± 3.08 and 31.02 ±
3.55, respectively (Table 2). The GSH content is 1.4-fold
higher in the HepG2 cells than in the Chang cells (Table
2). However, cancerous liver tissue that was removed
from patients contained higher GSH content than those in
the HepG2 cells (Huang et al., 2001). Although the GSH
content varies (dependent on cell sources), it clearly is
higher in hepatoma ceOOV -Glutamylcysteine synthetase
activity was determined to be 0.12 ± 0.10 and 0.72 ± 0.10
in the Chang and HepG2 cells, respectively (Table 2). The
HepG2 cells contained 5.9-IROGKLJKHU-glutamylcysteine
synthetase activity than the Chang cells. Glutathione synthetase activity was detected to be 0.07 ± 0.02 and 0.53 ±
0.16 in the Chang and HepG2 cells, respectively (Table 2).
Chang
Glutathioneb
-Glutamylcysteine synthetasec
Glutathione synthetasec
Glutathione reductasec
-Glutamyltranspeptidased
2 2 .7 4
0 0 .1 2
0 0 .0 7
0 0 .1 2
0 0 .0 3
HepG2
± 3 .0 8 (1 0 0 ) 3 1 .0 2 ± 3 .5 5 (1 4 0 )*
± 0 .1 0 (1 0 0 ) 0 0 .7 2 ± 0 .1 0 (5 9 0 )* *
± 0 .0 2 (1 0 0 ) 0 0 .5 3 ± 0 .1 6 (8 1 0 )* *
± 0 .0 2 (1 0 0 ) 0 0 .5 6 ± 0 .1 6 (4 9 0 )* *
± 0 .0 1 (1 0 0 ) 0 0 .3 1 ± 0 .0 7 (9 5 0 )* *
a
Values represent mean ± SD. Numbers in parentheses indicate relative specific activities.
b
*OXWDWKLRQHOHYHOZDVUHSUHVHQWHGDVJPJSURWHLQ
c
Activities of glutathione-synthesizing enzymes were represented
as ∆A340/min/mg protein.
d
-Glutamyltranspeptidase activity was represented as ∆A410/min/
mg protein.
*, p < 0.05 ; **, p < 0.001.
The HepG2 cells contained 8.1-fold higher activity in
glutathione synthetase activity than the Chang cells.
These results show that glutathione-synthesizing enzymes
are up-regulated in hepatoma cells. A similar phenomenon was previously shown in cancerous liver tissue from
patients by measuring their mRNA levels (Huang et al.,
2001). Glutatione reductase activity, which reduces oxidized GSH, was 0.12 ± 0.02 and 0.56 ± 0.16 in the Chang
and HepG2 cells, respectively, which indicates that the
HepG2 cells contained 4.9-fold higher activity (Table 2).
7KHHQ]\PH-glutamyltranspeptidase catalyzed the transIHU RI WKH -JOXWDP\O PRLHW\ RI -glutamyl-containing
compounds, notably GSH, to acceptors, including amino
acids, dipeptides, and GSH LWVHOI -Glutamyltranspeptidase activity was 0.03 ± 0.01 and 0.31 ± 0.07 in the
Chang and HepG2 cells, indicating that the HepG2 cells
contained much higher (9.5-IROG -glutamyltranspeptidase activity (Table 2). Therefore, GSH levels, GSHsynthesizing enzymes, and two glutathione-metabolizing
enzymes are significantly enhanced in the human hepatoma cell line HepG2.
GST is a member of a family of detoxification enzymes
that metabolize a variety of carcinogens by conjugating
lipophilic electrophiles to GSH. GST activity was 0.15 ±
0.01 and 0.05 ± 0.01 in the Chang and HepG2 cell lines,
respectively, indicating that the Chang cells contain much
higher activity (3.2-fold) (Fig. 1A). Decreased expression
of glutathione S-transferase π was reported to occur in the
human epidermoid cancer cell line (Yokomizo et al.,
1995). Nitrite contents in the Chang and HepG2 cell lines
appeared to be similar (Fig. 1B).
Thioltransferase (glutaredoxin), a small and ubiquitous
GSH-dependent disulfide oxidoreductase, participates in a
pathway that couples NADPH oxidation to the reduction
Yuk-Young Lee et al.
A
309
Table 3. Thioltransferase and thioredoxin reductase activities in
Chang and HepG2 cell lines.
Specific activitiesa
Enzymes
Chang
Thioltransferaseb
Thioredoxin reductasec
*
HepG2
1.70 ± 0.19 (100) 2.05 ± 0.18 (120)*
0.69 ± 0.07 (100) 0.22 ± 0.03 (32)*
a
Values represent mean ± SD. Numbers in parentheses indicate
relative specific activities.
b
Thioltransferase activity was represented as ∆A340/min/mg protein.
c
Thioredoxin reductase activity was represented as ∆A412/min/mg
protein.
*, p < 0.001.
B
Fig. 1. Levels of glutathione S-transferase activity (A) and nitrite content (B) in the Chang and HepG2 cell lines. Specific
activity is represented as ∆A340/min/mg protein. Nitrite content
is represented as ∆A543/mg protein. *, p < 0.001.
of ribonucleotide, sulfate, methionine sulfoxide, and arsenate (Holmgren, 1989). It may also be involved in cellular
responses to oxidative stresses. Thioltransferase activity
was 1.70 ± 0.19 and 2.05 ± 0.18 in the Chang and HepG2
cells, respectively (Table 3). It shows a 20% increase in
the HepG2 cells, which may help the survival and growth
of cancer cells. Thioredoxin reductase activities, which
belong to a family of glutathione reductase-like homodimeric flavoenzymes, were 0.70 ± 0.07 and 0.22 ± 0.03 in
the Chang and HepG2 cells, respectively (Table 3). Unexpectedly, thioredoxin reductase activity appeared to be
about 2-fold higher in the human normal hepatic cell line.
The human cell lines that were used in this study were
selected for two reasons: 1) Both of the cell lines were
derived from human liver tissue; therefore, they should
have the original phenotypes. 2) The Chang cell line is
normal; whereas, the HepG2 cell line is cancerous. By
comparing the levels of the defense system in the two cell
lines, we could estimate the changes that are due to tumorigenesis.
Reactive oxygen is related to both the growth arrest and
start of cell differentiation. Low concentrations of reactive oxygen intermediates may be beneficial, or even indispensable, in processes such as intracellular messaging
and defense against microorganisms. However, higher
amounts of active oxygen may be harmful to cells and
organisms (Matés and Sánchez-Jiménez, 1999). Antioxidant enzyme levels are important in both normal and tumor cells, and regulated in a very complicated manner. In
HepG2 cells, the overexpression of cytochrome P450 2E1
induces the catalase expression (Marí and Cederbaurn,
2001). This induction confers resistance to the cells
against several prooxidants. It is suggested to reflect an
adaptive response by the cells against cytochrome P450
2E1-mediated oxidative stress (Marí and Cederbaurn,
2001). Our data shows that the HepG2 cells already contain about 4-fold higher catalase activity than the Chang
cells. This may suggest that HepG2 becomes more resistant to oxidative stress. Furthermore, this may aid the survival of HepG2 cells. On the contrary, a reduction in catalase has been reported to correlate with the emergence of
the malignant phenotype, which suggests that attenuation
of catalase activity may play a functional role in the malignant progression of mouse keratinocytes (Gupta et al.,
2001). The other antioxidant enzymes (such as superoxide
dismutase and glutathione peroxidase) are greatly upregulated in HepG2 cells. Therefore, the HepG2 cell that
contains stronger antioxidant systems could easily defend
against reactive oxygen species that are produced during
its metabolic pathways. However, one report has shown
that increased manganese-containing superoxide dismutase decreases cell proliferation in various cell lines
(Oberley et al., 1995). The mechanism for the simultaneous enhancement of three antioxidant enzymes in HepG2
cells remains unclear.
GSH is the main non-protein thiol in mammalian cells.
It participates in many important cellular functions, such
as antioxidant defense and cell growth. The first step of
GSH biosynthesis is regarded as a rate-limiting step and
FDWDO\]HGE\-glutamylcysteine synthetase. Expression of
310
Antioxidant and Redox Enzymes in Human Cell Lines
WKH KXPDQ -glutamylcysteine synthetase is induced by
VRGLXP QLWURSUXVVLGH DQG -naphthoflavone (Galloway et
al., 1999; Mulcahy et al., 1997). Thioacetamide treatment
of the human normal hepatic cell line Chang was reported
to increase GSH and the glutathione synthetase expression (Huang et al., 2000) 7KH -glutamylcysteine synthetase is strongly expressed in nonsmall cell lung carcinomas and probably takes part in the defense of tumor
cells against oxidative damage (Soini et al., 2001). Cancer
cells become more sensitive to arsenic trioxide, an anticancer agent after depletion of cellular GSH with Lbuthionine sulfoximine (Yang et al., 1999). The cellular
GSH level is the most important determinant of arsenic
sensitivity in cancer cells. It was also demonstrated that
changes in the GSH content regulate the metastatic behavior of B16 melanoma cells (Carretero et al., 1999). GSH
has dual roles in the effects of selenite on the HepG2 cell.
These roles are as follows: 1) GSH acts as a pro-oxidant
that facilitates selenite-induced oxidative stress. 2) GSH
acts as an antioxidant that protects against seleniteinduced oxidative stress and apoptosis (Shen et al., 2000).
The present finding that GSH content is higher in the
HepG2 cells than in the Chang cells corresponds with the
previous result of increased GSH in lung carcinoma (Soini et al., 2001). The increased GSH amount in HepG2
occurs via the increased GSH-synthesizing enzymes, although the increase in GSH content does not clearly reflect the degree of increase in GSH-synthesizing enzymes.
GSH synthesis in HepG2 cells may depend on other factors. Otherwise, increased GSH may be consumed by
JURZWK UHTXLUHPHQW RU LQFUHDVHG -glutamyltranspeptidase. Higher levels of GSH content and its synthetic
enzymes may make HepG2 cells grow more safely in
various environments. However, the physiological meaning of decreased glutathione S-transferase and thioredoxin
reductase in HepG2 cells still needs to be elucidated. Thioredoxin reductase was previously found to be induced in
several tumors, which indicates that the thioredoxin system makes an important contribution to the resistant phenotype of the neoplastic liver cell (Bjorkhem et al., 2001).
Throughout this investigation, the defense enzyme levels
increased in the human hepatoma cell line HepG2. They
would be the targets for the development of anti-cancer
drugs.
Acknowledgments This work was supported by a research
grant (2001) from the Korea Sanhak Foundation, and carried out
by using facilities of Research Institute for Life Sciences at
KNU.
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