From the Departments of Biochemistry and
¶ Microbiology and Immunology, Kagoshima University Dental School,
Kagoshima 890-8544, Japan
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ABSTRACT |
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In this study, we show that
N-acetylcysteine (NAC), a precursor of glutathione and an
intracellular free radical scavenger, almost completely prevented
hepatocyte growth factor (HGF)-suppressed growth of Sarcoma 180 and
Meth A cells, and HGF-induced apoptosis, assessed by DNA fragmentation,
and increase in caspase-3 activity, in Sarcoma 180 cells. The reduced
form of glutathione also prevented HGF-suppressed growth of the cells
as effective as NAC. Ascorbic acid partially prevented the effect of
HGF, but other antioxidants such as superoxide dismutase, catalase, and
vitamin E, and the free radical spin traps
N-t-butyl- Hepatocyte growth factor
(HGF),1 which was initially
isolated as a potent mitogen of hepatocytes in primary culture (1, 2),
is now known to be a broad-spectrum mitogen of a variety of cell
types including melanocytes and endothelial and epithelial cells (3).
HGF has been found to be identical with the "scatter factor" (4),
which dissociates and increases the motility of epithelial cells. In
addition to these activities, HGF induces branching tubule formation of
Madin-Darby canine kidney epithelial cells in a three-dimensional
collagen gel matrix (5). HGF is a potent angiogenic factor in
vivo (6, 7) and is involved in organ regeneration (8) and
tumorigenesis (9). All these effects of HGF are mediated by the HGF
receptor, identified as the product of the c-met
proto-oncogene (10, 11). During embryogenesis, c-met is
expressed in epithelial components of various organs, while
hgf is expressed in the adjacent mesenchyme (12). Embryos of
mutant mice homozygous for the hgf gene die due to abnormal development of the placenta or both the placenta and liver (13, 14).
Likewise, c-met-deficient embryos lack muscles of the limbs, diaphragm, and tip of the tongue, all derived from migratory precursors (15). Thus, HGF is now understood as a multifunctional cytokine that
mediates epithelial-mesenchymal interactions.
In addition to its mitogenic activity, HGF has tumor cytotoxic
activity, which suppresses growth of several tumor cell lines including
Sarcoma 180 cells, Meth A mouse sarcoma cells, and Hep G2 human
hepatoblastoma cells (16-18). Moreover, HGF has been shown to inhibit
the growth of hepatocellular carcinoma cells in vivo and
c-myc-induced hepatocarcinogenesis in a transgenic mouse
model co-expressing c-myc and hgf (19-21).
However, little is known about the signaling pathway for the tumor
cytotoxic activity of HGF. Recently, we found that HGF activates the
apoptosis signaling pathway by increasing the activity of caspase-3, a
member of the interleukin 1 Reactive oxygen species (ROS) such as H2O2 are
produced during many physiological intracellular reactions and in
response to external stimuli such as UV radiation. They are, in
general, considered to be cytotoxic, but several lines of evidence
indicate that regulation of the intracellular redox status is a
versatile control mechanism in gene expressions and signal
transductions, including those mediating apoptosis (23, 24). Tumor
necrosis factor- The aim of this study was to evaluate the possible implication of
oxidative stress in the mechanism by which HGF induces tumor cell
killing. The present data provide direct evidence that HGF enhances the
production of ROS in Sarcoma 180 cells, and suggest that this is a
cause of the tumor cytotoxic activity of HGF.
Reagents--
The sources of materials used in this work were as
follows: 3-(4,5-dimethyl thiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) from Research Organics, Inc., Cleveland, OH; reduced form of
glutathione (GSH), N-t-butyl- Assay of Tumor Cytotoxic Activity--
The tumor cytotoxic
activity of HGF was determined by an MTT assay (34). Sarcoma 180 cells
(2 × 105 cells/ml) were plated on Nunc 24-well
plastic dishes (Roskilde, Denmark) in 0.4 ml of Dulbecco's modified
Eagle's medium supplemented with 10% fetal calf serum (basal medium)
and cultured as described previously (22). After 24 h, the cells
were preincubated with antioxidants for 1 h at 37 °C, then
cultured with or without HGF at the indicated concentrations for
48 h. Because NAC itself is a reductant and interferes with the
MTT assay, incubated medium with or without NAC was removed after
incubation and then 400 µl of basal medium containing MTT (0.5 mg/ml)
was added. This procedure almost completely rules out the reducing
effect of NAC in the MTT assay. After 30 min, this medium was removed,
formazan products were solubilized in 400 µl of acidified isopropyl
alcohol (0.04 N HCl in isopropyl alcohol) and the
absorbance of the MTT formazan products was measured at 570 nm using a
reference wavelength of 650 nm. For Meth A cells, the cells
(104 cells/ml) were plated on 24-well plastic dishes in 0.4 ml of RPMI 1640 (ICN Biomedicals, Aurora, OH) supplemented with 10% fetal calf serum and cultured with or without NAC in the presence or
absence of HGF as described above for 96 h. After incubation, the
cells were transferred to an Eppendorf tube and precipitated by
centrifugation. The cells were washed once with 1 ml of NAC-free medium
by centrifugation, and the MTT assay was performed as described above.
Preparation of DNA Fragments--
Cells were cultured as
described above except for use of 10 ml of medium/plate in 8.5-cm
plastic plates (Nunc). After incubation, the cells were collected and
washed, and about 5 × 106 cells were lysed in 200 µl of 10 mM Tris-HCl buffer (pH 7.5) containing 10 mM EDTA, 0.5% Triton X-100, and 0.1% SDS for 10 min at
4 °C as described previously (22). The lysate was centrifuged at
12,000 × g for 20 min, and the supernatant containing
fragmented DNA was treated with RNase A (100 µg/ml) for 60 min at
37 °C, and then digested with proteinase K (100 µg/ml) for 60 min
at 37 °C. Fragmented DNA was precipitated with isopropyl alcohol, washed with 70% ethanol and solubilized in 50 µl of 10 mM Tris-HCl (pH 7.5) containing 1 mM EDTA. Then
10-µl samples were subjected to electrophoresis in 1.2% agarose
gel containing 0.1 µg/ml ethidium bromide.
Assay of Caspase-3 Activity--
Caspase-3 activity was measured
essentially as described by Enari et al. (35). Briefly,
after incubation of cells in 8.5-cm plastic plates with or without
additives as indicated, they were collected, washed as described above,
and homogenized in 0.5 ml of extraction buffer (10 mM
HEPES-KOH, pH 7.4, 2 mM EDTA, 0.1% CHAPS, 5 mM
dithiothreitol, 100 µM
p-(amidinophenyl)methanesulfonyl fluoride hydrochloride, 10 µg/ml leupeptin, 1 µg/ml pepstatin A) in a Dounce homogenizer (type
A pestle). The cell extracts (80-90 µg of protein) obtained as
described above were diluted with 1 ml of reaction buffer (50 mM HEPES-KOH, pH 7.4, 10% sucrose, 0.1% CHAPS, 10 mM dithiothreitol, 0.1 mg/ml ovalbumin), and incubated for
30 min at 30 °C with 10 µM fluorescent substrate,
Ac-DEVD-MCA, in the presence or absence of 10 µM of a
specific inhibitor of caspase-3, Ac-DEVD-H. The fluorescence of the
cleaved substrate was determined with a spectrofluorometer (Type 850 Hitachi spectrofluorometer, Hitachi, Tokyo) at an excitation wavelength
of 380 nm and emission wavelength of 460 nm. Caspase-3-like activity
was determined by subtracting the values obtained in the presence of
inhibitor. One unit of enzyme activity corresponds to the activity that
cleaves 1 pmol of the substrate at 30 °C in 1 min/mg of protein.
Measurement of Intracellular ROS by Flow Cytometry--
A
peroxide-sensitive fluorescent probe DCFH-DA (36) was used to assess
levels of net intracellular generation of ROS. Cells treated with HGF
for the indicated times were incubated with 5 µM DCFH-DA
for 30 min. After incubation, cells were washed once with
Mg2+- and Ca2+-free phosphate buffered-saline,
detached by trypsinization, collected by centrifugation as described
above, and suspended in ice-cold phosphate-buffered saline. Levels of
intracellular ROS were measured with a FACScan (Becton Dickinson,
Mountain View, CA). In each analysis, 10,000 events were recorded.
Assay for Tyrosine Kinase Activity of c-Met--
After
preincubation of Sarcome 180 cells (2 × 105/ml) in
8.5-cm plastic plates with or without 5 mM NAC for 1 h, they were incubated with 30 ng/ml HGF for 5 or 10 min. After
incubation, they were collected, washed, lysed, resolved by
SDS-polyacrylamide gel electrophoresis, and subjected to Western
blotting analysis as described previously (22). Membranes were probed
with anti-phosphotyrosine or anti-mouse c-Met antibodies, and located
with an ImmunoStar kit (Wako Pure Chemical Industrials, Osaka),
according to the manufacturer's instructions.
Assay for Scatter Activity--
The scatter activity was assayed
according to Stoker et al. (37). Briefly, MDCK cells were
seeded at a density of 2 × 104 cells/ml of
Dulbecco's modified Eagle's medium, containing 10% fetal calf serum
in a 24-well plastic dish (0.4 ml/well), and they were allowed to
attach for 24 h. The cells were preincubated with or without NAC
(5 mM) for 1 h, and then incubated with or without HGF
(30 ng/ml). The cells were further incubated for 24 h.
Other Methods--
Protein was determined with BCA Protein Assay
Reagent (Pierce, Rockford, IL). Levels of the reduced form of
glutathione were determined in cell extracts by the method of Hissin
and Hilf (38), measuring the fluorescence produced by
o-phthalaldehyde as fluorescent reagent at an excitation
wavelength of 350 nm and emission wavelength of 420 nm. Student's
t test for unpaired samples was used to compare mean values
of groups according to Snedecor and Cochran (39).
Prevention of HGF-suppressed Growth of Sarcoma 180 Cells and Meth A
Cells by Antioxidants--
To study the role of oxidative stress on
the tumor cytotoxic activity of HGF, we first examined the effect of
the glutathione precursor NAC, which is widely used as an intracellular
free radical scavenger and has been shown to prevent diverse cell
death-inducing stimuli (30, 31), on HGF-suppressed growth of Sarcoma
180 cells. As described previously (22), incubation of the cells with
HGF (30 ng/ml) for 48 h resulted in reduction of viability to
about 50% of that of control cells (Fig.
1). As shown in Fig. 1A, when
the cells were incubated with NAC, the HGF-suppressed growth was
restored to nearly the control level. NAC was effective at 1 mM and 5 mM NAC restored HGF-suppressed
viability to about 85% of that of control cells. NAC itself (over 2 mM) had weak cytotoxic activity on the cells and 5 mM NAC decreased the viability about 16%. These results
indicate that NAC completely prevented HGF-suppressed growth of the
cells. In addition, we found that NAC did not shift the concentration
of HGF required for growth suppression. As shown in Fig. 1B,
100 ng/ml HGF decreased the viability of the cells about 60% and the
growth suppression restored to the control level by NAC treatment. The
preventive effect of NAC was also confirmed by cell counts. When the
cells were incubated with HGF (30 ng/ml) for 48 h, cell numbers
decreased to about 65% of that of control cells but the cell numbers
again were restored to the control level by NAC (5 mM)
treatment. Recently, HGF has been shown to induce apoptosis of Meth A
cells (40). Therefore, we examined whether NAC blocks HGF-induced
growth suppression of Meth A cells as Sarcoma 180 cells. As shown in
Fig. 1C, HGF caused reduction in the viability of the cells
about 30%, and NAC (5 mM) almost completely restored the
HGF-suppressed viability.
Other antioxidants and free radical trappers, e.g. GSH,
ascorbic acid, superoxide dismutase, catalase, vitamin E, PBN, and TEMPO, are also reported to prevent cell death induced by several cell
death-inducing stimuli (30, 31, 41-43). Therefore, we next examined
the effects of these compounds on HGF-suppressed growth of the cells.
As shown in Fig. 2, GSH prevented the
effect of HGF in a dose-dependent manner as effectively as
NAC. Ascorbic acid partially prevented the effect of HGF, but other
compounds at the concentrations tested did not have protective effects. PBN and TEMPO had cytotoxic effects on the cells. When the cells were
incubated with 5 mM NAC for 48 h, the intracellular
GSH level increased and the mean GSH contents (n = 2)
were 5.4, 12.1, and 11.1 µg/mg protein, in cell extracts without NAC,
with NAC, and with NAC plus HGF (30 ng/ml), respectively. These results
suggest a role of intracellular GSH in the protective effect of NAC
against HGF-induced growth suppression.
Prevention of HGF-induced Morphological Changes and Apoptosis in
Sarcoma 180 Cells by NAC--
HGF also caused morphological changes of
the cells (Fig. 3). Sarcoma 180 cells
without HGF had a flattened morphology during culture (Fig.
3A). But when they were incubated with HGF (30 ng/ml) for
48 h, many cells became condensed and rounded with an irregular plasma membrane (Fig. 3B). Detachment of degenerated cells
was always observed. These HGF-induced morphological changes were completely inhibited by co-incubation with 5 mM NAC (Fig.
3C), the cells showing a normal morphology, indicating the
involvement of oxidative stress in HGF-induced morphological
changes.
Previously, we found that HGF activates apoptosis in the cells, as
assessed by DNA fragmentation and an increase in caspase-3 activity
(22). As shown in Fig. 4A,
incubation of the cells with HGF (30 ng/ml) for 48 h resulted in
the formation of high molecular weight DNA fragments of about 20 kilobase pairs, as well as oligonucleosomal DNA fragments. HGF also
increased caspase-3 activity to about 2.4 times the control value after
48 h incubation (Fig. 4B). Both HGF-induced DNA
fragmentation and caspase-3 activity were almost completely inhibited
by NAC (5 mM), again establishing a role of oxidative
stress in regulating HGF-induced apoptosis. The results also indicated
that HGF-increased caspase-3 activity may be mediated directly or
indirectly by a redox-sensitive pathway, as suggested by others (44,
45).
Enhancement of Formation of Intracellular ROS in Sarcoma 180 Cells
by HGF--
We next investigated the effect of HGF on the formation of
intracellular ROS by flow cytometric analysis using a
peroxide-sensitive fluorescent probe DCFH-AD (36). This probe primarily
measures hydrogen peroxide and the hydroxy radical. As shown in Fig.
5A, HGF caused an increase in
the intracellular ROS level within 7 h incubation and increase in
the intracellular ROS level was completely suppressed by co-incubation
with 5 mM NAC (Fig. 5B). Increase in ROS was
also observed at 24 h (Fig. 5C) and 48 h (data not shown) after HGF treatment. NAC suppressed this increase (Fig. 5D, data not shown for 48 h).
Effect of NAC on HGF-stimulated Scattering of MDCK
Cells--
Finally, we investigated whether NAC prevents the
HGF-induced scattering (motility) of MDCK cells, which is one of well
known biological activities of HGF (4, 8). As shown in Fig.
6B, HGF treatment induced
scattering in MDCK cells after 24 h of incubation. However, NAC
did not prevent the HGF-induced scattering (Fig. 6C). NAC
itself had no affect the scattering of the cells (Fig. 6D).
HGF has diverse biological and physiological functions including
mitogenic, motogenic (scatter factor activity), morphogenic, and tumor
cytotoxic activities (8). To date, however, many studies have been
focused on understand the signaling pathways of HGF in mitogenic,
motogenic, and morphogenic activities (46-49), but little is known
about the mechanism of the signaling pathway in the tumor cytotoxic
activity of HGF. In this study, we obtained the first direct evidence
that HGF enhances the production of ROS in Sarcoma 180 cells (Fig. 5).
The present finding that the antioxidant NAC, which increases GSH,
thereby providing a substrate for the reduction of peroxide by
glutathione peroxidase (50), almost completely prevented HGF-induced
growth suppression, morphological changes, apoptosis, and ROS
production, suggests the involvement of oxidative stress in the tumor
cytotoxic activity of HGF.
HGF is a heterodimeric protein consisting of heavy and light chains
with molecular masses of 54-65 and 31.5-34.5 kDa, respectively, linked together by a disulfide bond (2) and its biological activity was
disappeared when it was reduced with reducing agents such as
2-mercaptoethanol (51). Because NAC is also a reductant, we examined
whether HGF was reduced by NAC during incubations, thereby destroying
HGF activity. When HGF (30 ng/ml) was treated with 5 mM NAC
in Dulbecco's modified Eagle's medium containing 10% fetal calf
serum at 37 °C for 48 h, the concentration of HGF decreased to
about 18 ng/ml (60% of the initial concentration), as determined by an
enzyme-linked immunosorbent assay which detects only the heterodimeric
form of HGF, but not the reduced form of HGF (52). However, the
decrease alone in the HGF content cannot explain all the protective
effects of NAC on HGF-induced growth suppression of Sarcoma 180 cells
reported in this paper, because of that (i) 10 ng/ml HGF decreased the
viability of the cells more than 40% (22) and (ii) 5 mM
NAC restored HGF-suppressed viability to about 85% of that of control
cells (Fig. 1). In addition, we showed that growth suppression of
Sarcoma 180 cells induced by 100 ng/ml HGF was also restored to the
control level by NAC treatment. In this condition, it can be presumed
that about 60 ng/ml HGF may still remain in the medium after 48 h
incubation with 5 mM NAC, as described above, and this
concentration of HGF should decrease the viability of the cells by at
least 50% of that of control cells (Fig. 3B). These
observations indicate that NAC does not shift the concentration of HGF
required for growth suppression and suggest that the effects of NAC on
the effects of HGF are mediated mostly through its antioxidant effect,
although it remains to be determined whether NAC affects directly other molecules such as HGF receptor c-Met or intracellular signal
transducing molecules. Interestingly, however, we found that NAC could
not prevent the HGF-induced scattering of MDCK cells (Fig. 6). These results further support the involvement of oxidative stress on tumor
cytotoxic activity of HGF. It also suggests the presence of other
mechanism(s) than oxidative stress on the scattering activity of HGF.
We recently found that HGF enhances expression of the
cyclin-dependent kinase inhibitor p21waf1,
cLip1, SDI1 within 7 h in Sarcoma 180 cells and that
this effect was not suppressed by 5 mM NAC
treatment.2 Thus, HGF seems
to activate diverse signaling pathways, NAC-sensitive and -insensitive
pathways, involved in growth suppression of the cells.
Extracellularly added superoxide dismutase and catalase have been shown
to protect against cell death induced by several stimuli inducing cell
death (30, 31, 41-43). However, these enzymes did not have protective
effects on HGF-induced growth suppression of Sarcoma 180 cells under
the conditions used (Fig. 2). Although these enzymes have been shown to
be taken up by liver cells by an endocytosis-dependent
mechanism (53, 54), one possible explanation for their inability to
prevent the effect of HGF is that their transports into Sarcoma 180 cells are limited. They may be instable in our system. The present
results also suggest that membrane lipid peroxidation is not involved
in HGF-induced growth suppression of Sarcoma 180 cells, since vitamin
E, a lipophilic free radical scavenger, showed no protection from the
effect of HGF.
Nitric oxide (NO) is a free radical species with multiple biological
functions and has been shown to be important in the cytotoxic activity
of macrophages on tumor cells and microbial pathogens (55-58). Because
NAC has protective effects on NO-mediated cytotoxicity and induction of
DNA single-strand breakage (57, 58) as well as oxidative damage, we
examined whether NO mediates HGF-induced cytotoxicity in Sarcoma 180 cells. NG-Monomethyl-L-arginine
monoacetate, which is a competitive inhibitor of NO synthases, and a NO
scavenger, carboxy-PTIO, were used to test the role of NO. However,
NG-monomethyl-L-arginine monoacetate
(up to 3 mM) and carboxy-PTIO (up to 100 µM)
did not have protective effects on HGF-suppressed growth of the cells,
although carboxy-PTIO itself slightly increased the viability of the
cells (data not shown), suggesting that NO is not involved in
HGF-induced growth suppression.
As defense systems, mammalian cells possess antioxidative enzymes
including three types of superoxide dismutase, catalase, glutathione
peroxidase, and glutathione S-transferase, which play roles
in protecting cells against oxidative damage. Kayanoki et al. (59) recently reported that transforming growth factor- HGF shows opposite biological activities in regulating cell death. It
induces growth suppression of some tumor cell lines (Refs. 19-22 and
this study), but prevents apoptosis induced by interferon--phenylnitrone and
3,3,5,5-tetramethyl-1-pyrroline-1-oxide did not have protective
effects. HGF caused morphological changes of the cells, many cells
showing condensation and rounding, and enhanced the generation of
intracellular reactive oxygen species (ROS) as judged by flow
cytometric analysis using 2',7'-dichlorofluorescein diacetate. NAC
completely prevented both HGF-induced morphological changes and the
enhancement of ROS generation in the cells. However, NAC did not
prevent the HGF-induced scattering of Madin-Darby canine kidney cells.
To our knowledge, this is the first report that HGF stimulates the
production of ROS, and our results suggest the involvement of oxidative
stress in the mechanism by which HGF induces growth suppression of
tumor cells.
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-converting enzyme family, without
affecting caspase-1 activity or the expressions of the apoptosis
suppressors Bcl-2 and Bcl-x in Sarcoma 180 cells (22). We also showed
that HGF-suppressed growth was partially restored when the apoptosis
signaling pathway was completely inhibited by a caspase-3 inhibitor.
This suggested the existence of other pathways, besides that for
induction of apoptosis, for the tumor cytotoxic activity of HGF.
(TNF-
) is known to induce apoptosis (25).
Recently, it was shown that TNF-
induces intracellular ROS and that
free radical scavengers inhibit TNF-
-mediated tumor cell killing
(26-28). In addition, many of the chemical and physical stimuli that
elicit programmed cell death also generate ROS such as
H2O2 and OH
(29). Low doses of
H2O2 induce apoptosis in a variety of cell types, again indicating a role of oxidative stress in regulating cell
death (23, 24). The involvement of oxidative stress is also suggested
in apoptosis induced by p53, transforming growth factor-
, and Fas
(30-32). Thus, it is likely that oxidative stress is a common signal
transducer for diverse cell death-inducing stimuli.
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-phenylnitrone
(PBN), 3,3,5,5-tetramethyl-1-pyrroline-1-oxide (TEMPO), ascorbic
acid, and superoxide dismutase from Sigma;
acetyl-Asp-Glu-Val-Asp-aldehyde (Ac-DEVD-H) and
Ac-DEVD-
-(4-methyl-coumaryl-7-amide) (Ac-DEVD-MCA) from the Peptide
Institute, Osaka, Japan; Dulbecco's modified Eagle's medium from
Nissui Pharmaceutical Co., Tokyo, Japan; Sarcoma 180 and Madin-Darby
canine kidney (MDCK) cells from RIKEN Cell Bank, Tsukuba, Japan; Meth A
mouse sarcoma cells were kindly provided by Dr. Eiichi Gohda, Faculty
of Pharmaceutical Sciences, Okayama University, Japan; DNA molecular
weight markers III and IX, and catalase from Roche Molecular
Biochemicals, Mannheim, Germany; 2',7'-dichlorofluorescein diacetate
(DCFH-DA) from Molecular Probes, Eugene, OR;
N-acetyl-L-cysteine (NAC),
NG-monomethyl-L-arginine
monoacetate,
2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxyde
(carboxy-PTIO), vitamin E (
-tocopherol), and
(p-amidinophenyl)methanesulfonyl fluoride hydrochloride from
Wako Pure Chemical Industrials, Osaka; monoclonal antibody PY20 against
phosphotyrosine and polyclonal antibodies against mouse c-Met from
Santa Cruz Biotechnology, Santa Cruz, CA. Recombinant human HGF was
prepared and purified as described previously (33). Other materials
used in this study were described previously (2, 22).
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Fig. 1.
Effect of NAC on HGF-induced growth
suppression of Sarcoma 180 (A and B)
and Meth A (C) cells. Cells were preincubated
with indicated concentrations (A) or 5 mM
(B and C) NAC for 1 h and then cultured with
30 ng/ml (A and C) or the indicated
concentrations (B) of HGF for 48 h (A and B)
or 96 h (C). Cell viability was analyzed by MTT assay
as described under "Experimental Procedures." Data are mean ± S.D. for three independent experiments. *, p < 0.01;
**, p < 0.001 (versus with HGF
alone).
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Fig. 2.
Effects of antioxidants on HGF-induced growth
suppression of Sarcoma 180 cells. Cells were preincubated with
antioxidants at the indicated concentrations for 1 h and then
cultured with (closed bars) or without (open
bars) 30 ng/ml HGF for 48 h. Viability of the cells was
analyzed as described in the legend to Fig. 1. Data are mean ± S.D. for three independent experiments. VE, vitamin E;
SOD, superoxide dismutase; *, p < 0.001;
**, p < 0.02 (versus value with HGF
alone).
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Fig. 3.
Morphological changes of Sarcoma 180 cells
induced by HGF. Cells were preincubated with (C and
D) or without (A and B) 5 mM NAC for 1 h, and then cultured in the presence
(B and C) or absence (A and
D) of 30 ng/ml HGF for 48 h. Then they were examined by
phase-contrast microscopy (magnification, × 200). Note that when the
cells were cultured with 30 ng/ml HGF for 48 h, many of them have
condensed and rounded showing an irregular shaped plasma membrane
(B), but that these morphological changes were inhibited by
co-incubation with 5 mM NAC (C). Similar results
were obtained in four independent experiments.
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Fig. 4.
Inhibition of HGF-induced apoptosis of
Sarcoma 180 cells by NAC. Panel A, cells were
preincubated with (lanes 3 and 4) or without
(lanes 1 and 2) 5 mM NAC for 1 h, and cultured with (lanes 2 and 3) or without
(lanes 1 and 4) 30 ng/ml HGF for 48 h. DNA
fragments were prepared, and analyzed as described in "Experimental
Procedures." Panel B, cells were preincubated with or
without 5 mM NAC for 1 h, and cultured in the presence
or absence of 30 ng/ml HGF for 48 h. Cell extracts were prepared
and caspase-3 activity was measured as described under "Experimental
Procedures." Values are mean ± S.D. for three independent
experiments. *, p < 0.001 (versus without
HGF); **, p < 0.002 (versus HGF
alone).
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Fig. 5.
Intracellular ROS production in Sarcoma 180 cells by HGF and its inhibition by NAC. Cells were preincubated
with (B and D) or without (A and
C) 5 mM NAC for 1 h, and cultured with
(black area) or without (white area) 30 ng/ml HGF
for 7 (A and B) or 24 h (C and
D). Cells were treated with the peroxide-sensitive
fluorescent probe DCFH-DA (5 µM) during the final 30 min
of each treatment. Relative peroxide concentrations in the cells were
then quantitated by flow cytometry. Representative results for four
experiments are shown.
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Fig. 6.
Effect of NAC on HGF-stimulated scattering of
MDCK cells. Cells were preincubated with (C and
D) or without (A and B) 5 mM NAC for 1 h, and then cultured in the presence
(B and C) or absence (A and
D) of 30 ng/ml HGF for 24 h. Then they were examined by
phase-contrast microscopy (magnification, × 100).
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suppresses the expressions of these antioxidative enzyme genes in adult
rat hepatocytes, thereby showing that production of ROS is increased in
transforming growth factor-
-treated cells. Therefore, we
investigated whether HGF affects the expressions of antioxidative enzymes by Western blotting and found that incubation of the cells with
HGF (30 ng/ml) for 7 h resulted in a decrease in the expression of
catalase to about 40% of that of control cells, but it did not affect
the expression of Cu2+, Zn2+-superoxide
dismutase (data not shown). When the cells were treated with 30 ng/ml
HGF for 7 h, glutathione peroxidase activity in the cell extracts
was also decreased to about 80% of that of control cells (data not
shown). Thus it seems likely that HGF induces intracellular ROS by
suppression of the expressions of some antioxidative enzymes in Sarcoma
180 cells. We are now examining the effects of HGF on the activities
and gene expressions of other antioxidative enzymes.
in
cultured mouse hepatocytes (60). The mechanism by which HGF exerts
these opposite effects is not known. In this regard, it has been
suggested that alterations in number or structure, with gain of
function, of growth factor receptors may result in deregulated
activation of the signaling pathway possibly involved in growth
suppression. An unusually high density of epidermal growth factor (EGF)
receptor in A431 cells has been suggested to be related to an increased
phosphotyrosine content and inhibition of proliferation of EGF-treated
cells (61, 62). In this connection, it is noteworthy that Bae et
al. (63) recently reported that EGF enhances the generation of
intracellular ROS in A431 cells, although the role of ROS in
EGF-suppressed growth of the cells has not been investigated. On the
other hand, mutations of fibroblast growth factor receptor 3 found in
human skeletal disorders (64) were shown to have constitutive tyrosine
kinase activity and expression of mutant fibroblast growth factor
receptor 3 in 293T cells caused activation of the transcription factor
STAT 1 (signal transducer and activator of transcription 1), which may
negatively regulate cell growth by inducing the
cyclin-dependent kinase inhibitor p21 (65). Like EGF and
fibroblast growth factors, all of the biological effects of HGF are
transduced through its receptor, Met, by the activation of its tyrosine
kinase activity (10, 11). Therefore, we examined tyrosine
phosphorylation of Met in the presence of HGF and NAC. However, we
found that Met in Sarcoma 180 cells was constitutively activated even
in the absence of HGF as mutant fibroblast growth factor receptor 3 and
EGF receptors in A431 cells (data not shown). Although treatment with
HGF alone for 5-10 min slightly activated (about 2-fold) its tyrosine
kinase activity in Sarcoma 180 cells, NAC had not affected the tyrosine phosphorylation of Met on the cells in the presence or absence of HGF
(data not shown). These results suggest that constitutive tyrosine
kinase activity of growth factor receptors is one common mechanism for
deregulated activation of the signaling pathway involved in growth
suppression, although HGF only slightly activated (about 2-fold) its
kinase activity. Further studies are necessary for complete
understanding of the mechanism by which HGF exerts its diverse
biological functions as well as on how HGF enhances intracellular ROS generation.
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ACKNOWLEDGEMENTS |
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We thank Dr. Takehisa Ishii, Mitsubishi Chemical Corp., for providing recombinant human HGF. We also thank Dr. Kyoko Kakimoto for determination of HGF and Yoko Amita for secretarial assistance.
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FOOTNOTES |
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* This work was supported in part by Grants-in-aid for a Priority Area (Cancer) and for Scientific Research from the Ministry of Education, Science, Sports and Culture of Japan.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ Present address: Faculty of Pharmaceutical Sciences, The University of Tokushima, 770-8505, Japan.
To whom correspondence should be addressed: Dept. of
Biochemistry, Kagoshima University Dental School, 35-1 Sakuragaoka-8, Kagoshima 890-8544, Japan. Tel.: 81-99-275-6130; Fax: 81-99-275-6138; E-mail: daiku{at}dentc.hal.kagoshima-u.ac.jp.
2 N. Arakaki, T. Kajihara, T. Ohnishi, and Y. Daikahara, unpublished observations.
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ABBREVIATIONS |
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The abbreviations used are:
HGF, hepatocyte growth factor;
ROS, reactive oxygen species;
TNF-, tumor
necrosis factor-
;
MTT, 3-(4,5-dimethyl thiazol-2-yl)-2,5-diphenyl
tetrazolium bromide;
GSH, reduced form of glutathione;
PBN, N-t-butyl-
-phenylnitrone;
TEMPO, 3,3,5,5-tetramethyl-1-pyrroline-1-oxide;
Asc, ascorbic acid;
Ac-DEVD-H, acetyl-Asp-Glu-Val-Asp-aldehyde;
Ac-DEVD-MCA, Ac-DEVD-
-(4-methyl-coumaryl-7-amide);
MDCK, Madin-Darby canine
kidney;
DCFH-DA, 2',7'-dichlorofluorescein diacetate;
NAC, N-acetyl-L-cysteine;
carboxy-PTIO, 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide;
NO, nitric oxide;
EGF, epidermal growth factor;
STAT, signal transducer and
activator of transcription;
CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid.
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REFERENCES |
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