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INTRODUCTION |
Vascular endothelial growth factor
(VEGF)1 is an important
regulator of the process of angiogenesis in many types of cancer, including ovarian cancer. High VEGF expression and microvessel density
have been correlated with poor survival in ovarian cancer patients (1).
VEGF exists as multiple isoforms that can be generated by alternative
splicing of a single transcript (2). VEGF121 is efficiently
secreted, whereas VEGF165 is partially secreted and partly
retained on the cell surface (2). Other variants, such as
VEGF145, VEGF189, and VEGF206,
remain primarily associated to the cell surface and to the
extracellular matrix (2). Expression of VEGF121,
VEGF145, VEGF165, and VEGF189
mRNAs has been detected in human ovarian cancer cell lines (3,
4).
Although the VEGF gene is controlled by different
transcription factors, the major regulator of its expression is
hypoxia-inducible factor 1 (HIF-1). HIF-1 is composed of two subunits,
HIF-1
and HIF-1
, that bind as a dimer to the hypoxia-responsive
element (HRE) in the VEGF promoter (5). HIF-1 mediates
activation of VEGF transcription in response to hypoxia in
solid tumors and in various malignant cell lines (5, 6). Multiple
stimuli, such as growth factors, hormones, nitric oxide, transition
metals, and iron chelators, can induce VEGF expression in a
HIF-1-dependent manner in normoxic cells (7-11).
The expression level of the HIF-1 protein is an important
determinant for the activity of HIF-1. Although HIF-1 protein expression is detected in the nucleus of normoxic cells, HIF-1 protein is undetectable in most cell types due to rapid degradation by
the ubiquitin-proteasome system (12-14). Hypoxia and many other activators of HIF-1 induce the accumulation of HIF-1 protein (9-11,
15). In hypoxic cells and in normoxic cells treated with transition
metals or iron chelators, HIF-1 protein levels are elevated as a
result of decreased ubiquitination and degradation (15). In some cases,
induction of HIF-1 protein expression may also involve an increase
in the rate of HIF-1 protein synthesis (16). In addition to its
level of expression, the HIF-1 protein is regulated at the level of
nuclear localization (17) and transactivation (15).
Recent studies suggest that alterations in the levels of reactive
oxygen species (ROS) provide a redox signal for HIF-1 induction by
hypoxia (18-20). Interestingly, ROS also appear to regulate HIF-1
activity under normoxia. In some cell types, increased ROS production
has been shown to mediate HIF-1 protein accumulation and
HIF-1-dependent transcription by hormones, growth factors, and transition metals (11, 19, 20). Moreover, direct exposure of cells
to ROS may also induce HIF-1 protein levels and/or VEGF expression (20, 21).
Up till now, it is not established whether modulation of
VEGF expression in tumor cells by ROS is a general
phenomenon. If this would be the case, cytotoxic agents in use for
cancer treatment might also up-regulate VEGF and, as a
consequence, VEGF downstream events (22, 23). Recent evidence indicates
that compounds containing trivalent arsenic (As3+,
arsenite) have potential as therapeutic agents in cancer (24). Exposure
to low dosages of arsenite in the form of arsenic trioxide (As2O3) and/or sodium arsenite
(NaAsO2) has a significant cytotoxic effect on different
malignant cell lines (24). Arsenic compounds are potent inducers of
oxidative stress, and evidence is provided that ROS may be mediators of
arsenite-induced cytotoxicity (25, 26).
To analyze whether oxidative stress can influence VEGF expression in
human ovarian cancer cells, we studied the effect of sodium arsenite
(NaAsO2). This agent has been shown to potently induce ROS
production in several cell systems (25, 26). Increased ROS production
may result from the activation of ROS-producing enzymes (25, 27) but
may also be associated with depletion of reduced glutathione (GSH).
Reduction of GSH levels by arsenite can be caused by the inhibition of
glutathione reductase (28). In addition, arsenite directly interacts
with thiol groups (SH) of GSH and cellular proteins (29-31).
Here, we report that arsenite induces VEGF expression in the human
ovarian cancer cell lines OVCAR-3 and H134. Arsenite-increased VEGF
expression was associated with the accumulation of HIF-1 protein in
both cell lines, suggesting a role for HIF-1 in this effect. By using
ROS inhibitors and thiol (anti)oxidants, we have assessed the possible
involvement of increased ROS production, GSH depletion, or direct
binding to thiol groups of cellular proteins in arsenite-induced VEGF
expression and HIF-1 protein accumulation.
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EXPERIMENTAL PROCEDURES |
Chemicals--
Sodium arsenite, actinomycin D, cycloheximide,
mannitol, catalase, GSH, N-acetylcysteine (NAC), and
buthionine-sulfoximine (BSO) were purchased from Sigma-Aldrich Chemie
(Zwijndrecht, The Netherlands).
Cell Culture and Cell Treatment--
The human ovarian cancer
cell lines OVCAR-3 and H134 were cultured in Dulbecco's modified
Eagle's medium (Dulbecco's modified Eagle's medium, Life
Technologies, Inc., Breda, The Netherlands) supplemented with 10%
heat-inactivated fetal calf serum (fetal calf serum, Sanbio, Uden, The
Netherlands), 50 units/ml penicillin (ICN Biochemicals, Zoetermeer, The
Netherlands), and 50 µg/ml streptomycin (ICN Biochemicals). Cells
were routinely cultured in 95% air and 5% CO2 at
37 °C. Hypoxic conditions were performed by incubation of cells in a
tightly sealed chamber maintained at 1% oxygen, 94% N2,
and 5% CO2 at 37 °C. Transient transfection of OVCAR-3
cells with pCEPHIF-1 (32) was performed by the calcium phosphate
precipitation method (33).
For treatment of cells with arsenite, cells were seeded in culture
dishes in medium and grown overnight. Thereafter, arsenite (100 or 30 µM) was added to the conditioned media, and cells were further incubated for the time periods as indicated in each experiment. Pretreatment of cells with actinomycin D (5 µg/ml) and cycloheximide (100 µM) was performed for 30 min and 2 h,
respectively, prior to the addition of arsenite (100 µM).
Pretreatment of cells with mannitol (50 and 100 mM),
catalase (500 and 1000 units/ml), GSH (10 and 20 mM), and
NAC (10 and 20 mM) were performed for 1 h prior to the
addition of arsenite, whereas pretreatment with BSO (500 µM) was performed for 16 h.
RNase Protection Assay--
The RNase protection assay was
carried out as described previously (34). The generation of the
-actin and VEGF165 antisense probes have been described
elsewhere (65). Hybridization to a 136-nt -actin antisense
probe gives rise to a protected fragment of 130 nt. Hybridization to a
301-nt VEGF165 antisense probe can give rise to fragments
of different sizes due to protection by VEGF mRNAs of different
isoforms. Hybridization of VEGF165 mRNA results in the
protection of a 252-nt fragment, whereas protection by
VEGF206/VEGF189 mRNAs or
VEGF145/VEGF121 mRNAs may give rise to
protected fragments of 170 and 82 nt, respectively (65). As protection
of the VEGF165 antisense probe by VEGF165
mRNAs is most efficient, effects on VEGF mRNA levels were
monitored by assessing the mRNA levels of VEGF165.
DNA templates for the synthesis of HIF-1 antisense probes were
generated by PCR. The expression vector pCEPHIF-1 (32) was used as a
template to amplify nt 1596-1831 of HIF-1 cDNA with the
forward primer
5'-AATTAACCCTCACTAAAGGGAGGATCAGACACCTAGTCCTT-3' and the
reversed primer
5'-GTAATACGACTCACTATAGGGCATCCATTGGGATATAGGGAG-3' for 36 cycles at 94, 56, and 72 °C. In addition to HIF-1
sequences (underlined), the forward and reverse primers contain
promoter sequences for T3 and T7 RNA polymerase, respectively. The
248-nt-amplified fragment was extracted with phenol/chloroform,
precipitated, and dissolved in T10E1. Use of
this 248-nt fragment as a template for the synthesis of a HIF-1
antisense probe with T7 RNA polymerase, resulted in a high background
signal in the RNase protection assay. To reduce the background signal,
the fragment was digested with EcoRI to generate a smaller
template of 125 nt with 5'-protruding ends. Using this template, a
125-nt HIF-1 antisense probe was synthesized with T7 RNA polymerase.
Because this antisense probe includes 119 nt of HIF-1 cDNA,
HIF-1 mRNA is expected to give rise to a protected fragment of
119 nt.
Preparation of Cell Extracts for ELISA and Western Blot
Analysis--
Cells were washed once with ice-cold phosphate-buffered
saline and lysed by scraping with a rubber policeman in 450 µl of E1A
buffer (50 mM Tris-HCl, pH 7.5, 250 mM NaCl, 5 mM EDTA, and 0.1% Nonidet P-40) for ELISA or 150 µl of
radioimmune precipitation buffer (10 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.1% SDS, 0.1% Nonidet P-40, and 0.1%
sodium deoxycholate) for Western blotting. Both lysis buffers were
supplemented with 50 mM NaF, 1 mM
Na3VO4, 1.0 mM phenylmethylsulfonyl
fluoride, 0.5 mM trypsin inhibitor, and 0.5 µg/ml
leupeptin. After a 15-min incubation period on ice, the extracts were
clarified by centrifugation at 14,000 rpm for 15 min at 4 °C and
stored at 
70 °C. Protein concentrations were determined by the
Coomassie Plus Protein assay (Pierce, Omnilabo, Breda, The Netherlands).
ELISA--
Equal numbers of cells were plated on nine 9.6-mm
culture dishes and grown overnight. The conditioned media of all dishes was collected and pooled, and 450 µl was sampled (T = 0 medium sample). Cells of one dish were washed and lysed in 450 µl
of lysis buffer as described above (T = 0 lysate
sample). The conditioned medium was divided in two equal volumes.
Arsenite was added to one volume at a concentration of 100 µM. Subsequently, conditioned media with or without
arsenite was again added to eight culture dishes with cells. Thus, at
T = 0 the amount of VEGF protein in the medium was the
same in each culture dish. After incubation periods of 2, 4, 6, and
8 h, 450 µl of conditioned medium was sampled and cells were
lysed. VEGF concentrations in nondiluted media samples and lysates were
determined in duplicate by ELISA using the reagents and the
protocol supplied with the Quantikine Human VEGF Immunoassay kit (R&D
Systems/ITK Diagnostics, Uithoorn, The Netherlands). Differences in
VEGF concentrations in medium and lysates of nontreated
versus arsenite-treated cells were evaluated using
Student's t test for two groups. p values <0.05
were considered to be significant.
Western Blot Analysis--
In the Western blot experiments, 100 µg of protein cell extract was resolved in a SDS-polyacrylamide gel
(7.5%) and electrophoretically transferred onto a polyvinylidene
difluoride membrane (Immobilon, Millipore, Etten-Leur, The
Netherlands). Membranes were blocked for 1 h in TBST (10 mM Tris, pH 8.0, 150 mM NaCl, and 0.025% Tween 20)/5% milk and incubated overnight at 4 °C with
HIF-1-directed antiserum in a 1:1000 dilution. After washing with
TBST, the membranes were incubated for 1 h with horseradish
peroxidase-linked anti-mouse antiserum in TBST/5% milk. The membranes
were washed again with TBST, and proteins were visualized by Electro
Chemoilluminescence. The mouse monoclonal antiserum to HIF-1
(H1alpha67, catalogue no. ab1-100) and the horseradish
peroxidase-coupled anti-mouse serum (catalogue no. P0260) were from
Novus Biologicals/AbCam (Cambridge, United Kingdom) and DAKO (Glostrup,
Denmark), respectively.
Measurement of ROS--
Intracellular ROS production was
assessed using 2',7'-dichlorofluorescein diacetate (DCFH-DA; Molecular
Probes, Leiden, The Netherlands). After diffusion into cells, this dye
is hydrolyzed by intracellular esterase to yield
2'-7'-dichlorofluorescein (DCFH). ROS (hydrogen peroxide or low
molecular weight peroxides) in the cells oxidizes DCFH to the highly
fluorescent compound 2',7'-dichlorofluorescein (DCF). Cells were plated
on culture dishes and incubated with arsenite (100 µM)
for different time periods. DCFH-DA was added to the medium 1 h
before harvesting the cells by trypsinization. Cell fluorescence was
detected with a FACScan flow cytometer using a 525-nm band pass filter.
The relative mean fluorescence intensity in arsenite-treated cells
after individual incubation periods (i) was tested against
100% (t = M(i) 100/S.E.) by means of Student's t test for one
group. The p values <0.05 were considered to be
significant. In addition, the relative mean fluorescence intensity in
cells treated with arsenite and catalase or mannitol (c,
m) was tested against the relative mean fluorescence
intensity in cells treated with arsenite alone or to control cells
(t = M(c, m) 100/S.E.) using the Student's t test for two groups and one
group, respectively. The p values <0.05 were considered significant.
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RESULTS |
Arsenite Induces VEGF mRNA and Protein Levels in OVCAR-3 and
H134 Cells--
VEGF mRNA levels in the human ovarian cancer cells
OVCAR-3 and H134 upon different periods of exposure to 100 µM arsenite were assessed by the RNase protection assay
(Fig. 1). In this assay, a labeled,
antisense VEGF165 RNA probe was used for hybridization, allowing efficient detection of mRNAs encoding the
VEGF165 isoform. Hybridization of total RNA of nontreated
OVCAR-3 and H134 cells resulted in the protection of a 252-nt fragment,
indicating a basal level of expression of VEGF165 in both
cell lines. Judged by the intensity of the 252-nt protected fragment,
the basal level of VEGF165 mRNA in H134 cells appeared
to be slightly higher than that in OVCAR-3 cells. After 2 h of
arsenite treatment, the intensity of the 252-nt fragment relative to
the -actin-protected fragment increased in both OVCAR-3 and H134
cells, indicating an increase in VEGF165 mRNA levels.
This increase was sustained until at least 8 h of exposure. At
this time point, induction of VEGF165 mRNA levels was
~6- and 3-fold in OVCAR-3 and H134 cells, respectively. Changes in
VEGF165 mRNA levels were not observed in nontreated cells (data not shown).
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Fig. 1.
Sodium arsenite induces VEGF165
mRNA levels in OVCAR-3 and H134 cells. OVCAR-3 and H134 cells
were exposed to sodium arsenite (100 µM) for the
indicated time periods. Total RNA was extracted and hybridized to
VEGF165 and -actin antisense probes in the RNase
protection assay. tRNA (T) was hybridized as a negative
control. The 252- and 130-nt fragments protected by the mRNAs of
VEGF165 and -actin and the full-length probes are
indicated.
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We next assessed whether induction of VEGF mRNA levels by arsenite
resulted in an increased production of VEGF protein. Arsenite was added
to the conditioned medium of OVCAR-3 and H134 cells, and VEGF
concentrations were measured by ELISA in the conditioned media or in
cell lysates after different periods of incubation. In addition, we
determined VEGF protein concentrations in conditioned media and lysates
of nontreated cells. As can be seen in Fig. 2A, VEGF protein levels in the
conditioned medium of H134 cells were much higher than in those of
OVCAR-3 cells at the start of treatment (T = 0). Upon
exposure to arsenite, a time-dependent increase in VEGF
production was observed in both cell types. Increased VEGF protein
levels in the conditioned media of arsenite-treated versus
nontreated cells was observed after 6 and 8 h of incubation. In
OVCAR-3 cells, this increase was statistically significant. In the
lysates, increased production of VEGF was evident and statistically significant after 4 h of incubation and was more pronounced after 6 and 8 h (Fig. 2B).
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Fig. 2.
Sodium arsenite induces VEGF protein levels
in OVCAR-3 and H134 cells. Sodium arsenite (100 µM)
was added to conditioned media of OVCAR-3 and H134 cells and at the
indicated time points the concentrations of VEGF protein in conditioned
media (A) and cell lysates (B) were measured by
ELISA as described under "Experimental Procedures." As a control,
VEGF protein concentrations were determined in conditioned medium and
lysates of nontreated cells. Results are given in picograms of VEGF per
milliliter of conditioned medium in (A) and per milligram of
total cell protein in B. The histograms represent the
mean ± S.D. of duplicate samples in a representative experiment
of three independent experiments that gave comparable results.
Significant differences in mean VEGF concentrations in medium and
lysates of nontreated versus arsenite-treated cells after
individual incubation periods are indicated by an asterisk
(p < 0.05).
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It should be noted that, in contrast to the RNase protection assay, the
ELISA assay does not discriminate between VEGF165 and the
other VEGF isoforms. Therefore, it is difficult to assess exact
correlations between VEGF mRNA and protein levels. Nevertheless, these data indicate that induction of VEGF mRNA upon arsenite treatment is associated with the accumulation of VEGF protein in H134
and OVCAR-3 cells and in their conditioned media.
Arsenite-induced VEGF Expression Is at the Transcriptional Level
and Is Dependent on de Novo Protein Synthesis--
To determine
whether induction of VEGF mRNA levels by arsenite was due to
increased transcription or to RNA stabilization, we analyzed the effect
of the transcription inhibitor actinomycin D in both OVCAR-3 and H134
cells. Fig. 3 shows that actinomycin D
completely inhibited induction of VEGF165 mRNA levels
by arsenite in both cell types, suggesting that this effect is at the
transcriptional level.
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Fig. 3.
Induction of VEGF165 mRNA by
sodium arsenite is at the transcriptional level. OVCAR-3 and H134
cells were exposed for the indicated time periods to sodium arsenite
(100 µM) in the absence or presence of actinomycin D (5 µg/ml) or to actinomycin D (5 µg/ml) alone. Actinomycin D was added
30 min prior to the addition of sodium arsenite. Total RNA was
extracted and hybridized to VEGF165 and -actin antisense
probes in the RNase protection. tRNA (T) was hybridized as a
negative control. The 252- and 130-nt fragments protected by the
mRNAs of VEGF165 and -actin and the full-length
probes are indicated.
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To analyze whether arsenite-induced expression of VEGF165
was dependent on de novo protein synthesis, we determined
the effect of the protein synthesis inhibitor cycloheximide. As can be
seen in Fig. 4A, induction of
VEGF165 mRNA levels upon arsenite treatment in OVCAR-3
cells was strongly inhibited by cycloheximide. In H134 cells,
VEGF165 mRNA levels were potentiated with cycloheximide alone. Incubation of cycloheximide-pretreated H134 cells with arsenite
did, however, not increase VEGF165 mRNA levels beyond those observed by cycloheximide alone. These data suggest that induction of VEGF expression by arsenite is dependent on ongoing protein synthesis.
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Fig. 4.
Induction of VEGF expression and
HIF-1 protein by sodium arsenite is dependent
on de novo protein synthesis. OVCAR-3 and H134
cells were exposed for the indicated time periods to sodium arsenite
(100 µM) in the absence or presence of cycloheximide (100 µM) or to cycloheximide (100 µM) alone.
VEGF mRNA levels and HIF-1 protein levels were assessed by RNase
protection (A) and Western blotting (B),
respectively. In the RNase protection experiment, total RNA was
hybridized to VEGF165 and -actin antisense RNA probes.
tRNA (T) was hybridized as a negative control. The 252- and
130-nt fragments protected by the mRNAs of VEGF165 and
-actin and the full-length probes are indicated.
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Arsenite-induced VEGF Expression Is Associated with the
Accumulation of HIF-1 Protein--
To examine whether
arsenite-induced VEGF expression may be mediated by HIF-1, we
investigated the HIF-1 protein levels upon arsenite treatment in
OVCAR-3 and H134 cells by Western blot with a HIF1-directed antibody
expression. As can be seen in Figs. 4B and 5A,
OVCAR-3 and H134 cells showed a basal level of HIF-1 protein
expression. After 4 h of arsenite treatment, the level of HIF-1
protein was elevated and was even further increased after 8 h.
Note that, under conditions where cycloheximide was found to inhibit
arsenite-induced VEGF mRNA levels, the accumulation of HIF-1
protein was inhibited over 90% in both OVCAR-3 and H134 cells (Fig.
4B).
We also assessed the effects of arsenite on the levels of HIF-1
mRNA by the RNase protection assay (Fig.
5B). Hybridization of total
RNA of nontreated OVCAR-3 cells to a HIF-1 antisense probe resulted
in the protection of a fragment with the expected size of 119 nt. As a
positive control, we also hybridized total RNA of OVCAR-3 cells that
were transiently transfected with a HIF-1 expression vector. As
expected, the 119-nt fragment was detected with increased intensity,
whereas it was not observed after hybridization with control tRNA.
These data confirm that the 119-nt fragment is protected by HIF-1
mRNA and that detection of this fragment is indicative for a basal
level of expression of HIF-1 mRNA in nontreated OVCAR-3 cells.
The intensity of the 119-nt fragment did not alter relatively to that
of the -actin-protected fragment until at least 8 h of exposure
to arsenite. The same results were obtained in H134 cells (data not
shown). These findings indicate that HIF-1 mRNA levels are not
influenced upon treatment with arsenite and that induction of HIF-1
protein is regulated by a post-transcriptional mechanism.
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Fig. 5.
Sodium arsenite induces the stabilization of
HIF-1 protein. A and
B, OVCAR-3 cells were exposed to sodium arsenite (100 µM) and protein, and total RNA was extracted at the
indicated time points. HIF-1 protein and mRNA levels were
assessed by Western blotting with a HIF-1-directed antiserum
(A) and the RNase protection assay (B),
respectively. In the RNase protection experiment, total RNA was
hybridized to a HIF-1 antisense RNA probe. As a positive control,
hybridization was performed with total RNA of OVCAR-3 cells that were
transiently transfected with an expression vector encoding human
HIF-1 (PC). tRNA (T) was hybridized as a
negative control. The 119- and 130-nt fragments protected by the
mRNAs of HIF-1 and -actin and the full-length probes are
indicated. C, HIF-1 expression was induced by exposure of
OVCAR-3 cells to 1% O2 (hypoxia (Hyp);
upper panel) for 6 h or to sodium arsenite (100 µM) for 5 h under normoxia (Norm;
lower panel). Cycloheximide (CHX) was added to a
final concentration of 100 µM, and cells were further
incubated under normoxia. Cell lysates were prepared at the indicated
time periods after cycloheximide addition. HIF-1 protein levels were
assessed by Western blotting with a HIF-1-directed antiserum. In
A and C, an unidentified protein that is
aspecifically recognized by the HIF-1-directed antiserum or
by the secondary horseradish peroxidase-linked antiserum is indicated
as NS.
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The low level of expression in nonstimulated OVCAR-3 and H134 cells
suggests that HIF-1 protein is unstable under normoxic conditions in
these cell lines. To assess whether arsenite regulates the expression
of HIF-1 protein by inhibiting its degradation, we monitored the
levels of arsenite-induced HIF-1 protein after blocking protein
synthesis by cycloheximide. First, we confirmed that HIF-1 protein
is rapidly degraded in the absence of protein synthesis in
nonstimulated OVCAR-3 and H134 cells under normoxia. To this end, we
investigated the decay of hypoxia-stabilized HIF-1 after transfer of
cells from hypoxia to normoxia. In agreement with previous studies in
other cell types (12, 13, 35), a rapid decay of HIF-1 protein was
observed within 15 min after transfer from hypoxia to normoxia in
OVCAR-3 and H134 cells (Fig. 5C and data not shown).
HIF-1 protein was completely undetectable by the end of 30 min under
normoxia. In contrast, arsenite-induced HIF-1 protein levels under
normoxia remained constant for 15 min after cycloheximide addition and
decreased relatively slowly to ~50% within 45 min thereafter.
Despite the lack of protein synthesis, the levels of HIF-1 protein
were still detectable and clearly above basal after 3 h of
cycloheximide exposure. The decay of arsenite-induced HIF-1 protein
in the absence of protein synthesis was found to occur with similar
kinetics in H134 cells (data not shown). Note that the effects
of arsenite and hypoxia in OVCAR-3 cells were specific for
HIF-1 protein, because the levels of a protein that was
nonspecifically recognized by the HIF-1 or secondary
antiserum remained constant under all conditions. These results
indicate that arsenite induces HIF-1 protein in normoxic OVCAR-3 and
H134 cells by slowing down its degradation.
The finding that increased levels of VEGF165 mRNA are
associated with the stabilization of HIF-1 protein in
arsenite-treated OVCAR-3 and H134 cells may be indicative for a role of
HIF-1 in the induction of VEGF expression by arsenite.
Arsenite-induced VEGF Expression and HIF-1 Protein Accumulation
Is Independent of ROS--
Arsenite has been reported to induce the
production of different types of ROS, including superoxide anions
(O
2), hydrogen peroxide (H2O2), and
hydroxyl radicals (OH·) (26). To investigate the role of ROS in
arsenite-induced VEGF expression and HIF-1 protein accumulation, we
first tested whether arsenite treatment increased the production of
H2O2 in OVCAR-3 cells. OVCAR-3 cells were
treated with 100 µM arsenite for different time periods,
and 80 µM DCFH-DA was added to the medium 1 h before cells were harvested by trypsinization. Intracellular peroxide levels
in trypsinized cells were monitored by flow-cytometric analysis. Fig.
6 shows that a gradual increase in
intracellular peroxide levels was detectable at the various time points
tested, which reached statistical significance after 8 h of
exposure.
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Fig. 6.
Sodium arsenite increases ROS production in
OVCAR-3 cells. A, OVCAR-3 cells were treated with
sodium arsenite (100 µM) for the indicated time periods.
DCFH-DA (60 µM) was added 1 h before harvesting of
cells by trypsinization. Fluorescence in the cells was determined using
flow cytometry. The mean fluorescence intensity (M1) is
indicated in the histograms. Induction of M1 by sodium arsenite varied
in different experiments from 2- to 5-fold after 8 h of exposure.
In the experiment shown, induction of M1 after 8 h of exposure was
~5-fold. The values in the bar graph are the mean ± S.E. of the relative fluorescence intensities from three independent
experiments in which nontreated controls were set at 100%. Significant
differences between the relative mean fluorescence intensities after
incubation with arsenite and that of control cells are indicated by an
asterisk (p < 0.05).
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To further investigate a possible relationship between the induction of
VEGF expression and HIF-1 protein and intracellular peroxide levels
in arsenite-treated OVCAR-3 cells, we tested the effect of the ROS
inhibitors catalase and mannitol. Catalase can inactivate hydrogen
peroxide by decomposing it into water and oxygen, whereas mannitol is a
selective scavenger of hydroxyl radicals. Because hydrogen peroxide can
function as a precursor of hydroxyl radicals (Fenton reaction) (26),
the scavenging of hydroxyl radicals by mannitol may also lead to a
reduction in the intracellular hydrogen peroxide levels. As shown in
Fig. 7A, pretreatment with
mannitol (50 or 100 mM) or catalase (500 or 1000 units/ml)
was found to significantly reduce arsenite-induced fluorescence
intensity to levels that were not significantly below those observed in
control cells. This indicates that induction of hydrogen peroxide
levels by arsenite was efficiently blocked. In contrast, induction of
VEGF165 mRNA and HIF-1 protein levels was hardly
affected by mannitol and catalase pretreatment (Fig. 7, B
and C). In the experiment presented in Fig. 7A,
9 h of exposure to mannitol (100 mM) or catalase (1000 units/ml) alone was also found to reduce the basal fluorescence
intensity of 40.5 (control, M1) to 29.1 and 9.9, respectively (histograms not shown). Both agents, however, did not
significantly influence the basal levels of VEGF165
mRNA and HIF-1 protein in OVCAR-3 cells. The concentrations of
mannitol and catalase that were used here were not toxic for OVCAR-3
cells and have been shown to be nontoxic in other in vitro studies. All together, these results strongly suggest that
arsenite-induced VEGF expression and HIF-1 protein accumulation is
independent of increased ROS production in OVCAR-3 cells.
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Fig. 7.
Induction of VEGF expression and
HIF-1 protein by sodium arsenite is not
mediated by increased ROS production. OVCAR-3 cells were treated
with sodium arsenite (100 µM) in the absence or presence
of mannitol (50 or 100 mM) or catalase (500 or 1000 units/ml). Mannitol and catalase were added 1 h prior to sodium
arsenite exposure. In addition, cells were treated with mannitol (100 mM) and catalase (1000 units/ml) alone. Effects of mannitol
and catalase on the basal and sodium arsenite-induced levels of
intracellular hydrogen peroxide, VEGF165 mRNA, and
HIF-1 protein were assessed by using DCFH-DH (A), RNase
protection (B), and Western blotting (C),
respectively. In A, DCFH-DH (60 µM) was added
7 h after the addition of sodium arsenite and cells were harvested
by trypsinization after 60 min. Fluorescence in the cells was
determined using flow cytometry. The mean fluorescence intensity
(M1) is indicated in the histograms. The results are
representative of three different experiments. The values in the
bar graph are the mean ± S.E. of the relative
fluorescence intensities of different treatments from three independent
experiments in which nontreated controls were set at 100%. Significant
differences in the relative mean fluorescence intensities after
treatment with arsenite alone or with arsenite in the presence of
catalase or mannitol is indicated by an asterisk
(p < 0.05). In B, total RNA was extracted
8 h after the addition of sodium arsenite and hybridized to
VEGF165 and -actin antisense probes. tRNA (T)
was hybridized as a negative control. The 252- and 130-nt fragments
protected by the mRNAs of VEGF165 and -actin and the
full-length probes are indicated. In C, cell extracts were
prepared 8 h after the addition of sodium arsenite.
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Induction of VEGF Expression and HIF-1 Protein by Arsenite Is
Modulated by Intracellular GSH Levels in OVCAR-3 Cells--
Previous
studies have shown that the level of intracellular GSH is an important
determinant for the biological effects of arsenite. Elevated levels of
intracellular GSH often antagonize the effects of arsenite, whereas
they are potentiated by depletion of intracellular GSH (28, 36, 37). To
examine the role of intracellular GSH levels in the induction of VEGF
mRNA levels and HIF-1 protein by arsenite in OVCAR-3 cells, we
tested the effect of pretreatment with GSH and NAC. The latter agent is
known to induce intracellular GSH levels by acting as a precursor for the synthesis of GSH. As shown in Fig.
8A, pretreatment with GSH inhibited induction of VEGF165 mRNA levels by arsenite
in a concentration-dependent manner. A partial inhibition
was observed with 10 mM GSH, whereas complete inhibition
was obtained at the 20 mM concentration. Induction of
VEGF165 mRNA levels was also completely abrogated by
pretreatment with 10 and 20 mM concentrations of NAC. The
inhibitory effect of GSH and NAC on VEGF165 mRNA
induction was associated with the inhibition of HIF-1 protein
accumulation (Fig. 8B). By themselves, GSH (20 mM) and NAC (20 mM) had only a minor effect on
VEGF165 mRNA and HIF-1 protein levels in OVCAR-3
cells.
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Fig. 8.
Induction of VEGF expression and
HIF-1 protein by sodium arsenite is inhibited
by GSH and NAC pretreatment. OVCAR-3 cells were exposed to sodium
arsenite (100 µM) in the absence or presence of GSH (10 or 20 mM) or NAC (10 or 20 mM). GSH and NAC
were added 1 h before the addition of sodium arsenite. OVCAR-3
cells were also treated with GSH (20 mM) and NAC (20 mM) alone. After 8 h of exposure to sodium arsenite,
total RNA was extracted and whole cell extracts were prepared. Effects
of GSH and NAC on basal and arsenite-induced VEGF165
mRNA and HIF-1 protein levels were assessed by RNase protection
(A) and Western blotting (B), respectively, In
A, total RNA was hybridized to VEGF165 and
-actin antisense probes. tRNA (T) was hybridized as a
negative control. The 252- and 130-nt fragments protected by the
mRNAs of VEGF165 and -actin and the full-length
probes are indicated.
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To obtain additional evidence for a role of GSH in arsenite-induced
VEGF expression and HIF-1 protein accumulation in OVCAR-3 cells, we
assessed the effect of pretreatment with BSO. BSO depletes the
intracellular GSH pool by inhibiting glutamylcysteine synthase, which
plays a critical role in the synthesis of GSH (38). Because arsenite is
known to reduce GSH levels (28), we speculated that potential effects
of GSH depletion would be more pronounced when cells were treated with
a suboptimal dose of arsenite (30 µM). As can be seen in
Fig. 9A, OVCAR-3 cells
displayed a slower, more gradual increase in VEGF165
mRNA and HIF-1 protein levels upon treatment with 30 µM arsenite. Pretreatment with BSO (500 µM) was found to potentiate the elevation of VEGF165 mRNA
as well as HIF-1 protein levels after 6 and 8 h of exposure to
arsenite (Fig. 9). Pretreatment with BSO alone (500 µM)
for a period of 24 h hardly affected the basal levels of
VEGF165 mRNA and HIF-1 protein in OVCAR-3 cells. In
summary, these results suggest that the intracellular GSH content is an
important determinant in the regulation of VEGF expression and HIF-1
protein levels by arsenite in OVCAR-3 cells.
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Fig. 9.
Induction of VEGF expression and
HIF-1 protein by sodium arsenite is
potentiated by BSO pretreatment. OVCAR-3 cells were exposed to a
suboptimal concentration of sodium arsenite (30 µM) in
the absence or presence of BSO (500 µM) or to BSO (500 µM) alone. BSO was added 16 h before the addition of
sodium arsenite. After the indicated periods of arsenite exposure,
total RNA was extracted and whole cell extracts were prepared. Effects
of BSO on basal and sodium arsenite-induced VEGF165
mRNA and HIF-1 protein levels were assessed by RNase protection
(A) and Western blotting (B), respectively. In
A, total RNA was hybridized to VEGF165 and
-actin antisense probes. tRNA (T) was hybridized as a
negative control. The 252- and 130-nt fragments protected by the
mRNAs of VEGF165 and -actin and the full-length
probes are indicated.
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DISCUSSION |
This study was undertaken to examine whether oxidative stress can
influence VEGF expression levels in human ovarian cancer cells. To this
end, we examined the effects of the oxidative stressor arsenite in the
two human ovarian cancer cell lines OVCAR-3 and H134. Arsenite was
found to induce VEGF mRNA and VEGF protein levels in both cell
lines. Our results suggest that this effect involves transcriptional
activation of the VEGF gene. First, elevation of VEGF
mRNA levels in arsenite-treated OVCAR-3 and H134 cells was
completely attenuated by the transcription inhibitor actinomycin D. Second, induction of VEGF mRNA levels was found to be associated with the accumulation of HIF-1 protein in both cell lines,
indicating that arsenite may induce VEGF expression through
the activation of HIF-1. This would be consistent with our results from
experiments with cycloheximide showing that arsenite-induced VEGF
expression is dependent on de novo protein synthesis.
It should be noted that, in both cell lines, an increase in VEGF
mRNA levels was detectable after 2 h of exposure to 100 µM arsenite, whereas clear elevation of HIF-1 protein
levels was not detected until 4 h of exposure (Figs. 1 and 5).
Consistently, OVCAR-3 cells that were treated with a suboptimal dose of
arsenite (30 µM) displayed increased VEGF mRNA levels
after 4 h of treatment, whereas induction of HIF-1 protein was
observed after 6 h (Fig. 9). Because a basal level of HIF-1
protein expression is detected in both cell lines, arsenite may
increase HIF-1 activity at earlier time points by affecting the nuclear
localization or the transcriptional activity of pre-existing HIF-1
protein. Alternatively, arsenite-induced VEGF expression may involve
the activation of other or additional transcription factors. A possible
candidate may be the transcription factor AP-1, which has been shown to
be activated by arsenite (37). The VEGF promoter contains
six potential binding sites for AP-1 that are located at positions
1892 to 1886, 1227 to 1221, 1129 to 1123, 937 to 931,
490 to 484, and +418 to +424 relative to the transcription start at
position +1 (5, 39, 40). Deletion/mutation analysis of the
VEGF promoter using luciferase-reporter constructs has
implicated AP-1 transcription factors in VEGF transcription
regulation. AP-1 binding to 1129 to 1123 and to 937 to 931
potentiates HIF-1-mediated induction of VEGF expression by
hypoxia in human glioma cell lines (10, 40). In one of those cell
lines, the AP-1 element at 937 to 931 was also shown to potentiate
HIF-1-dependent activation of VEGF by nitric
oxide (10). AP-1 may, however, also mediate induction of
VEGF expression in an HIF-1-independent manner (41, 42). Additional transcription factors that are involved in VEGF
regulation are SP-1 and AP-2, which mediate activation of
VEGF by various growth factors (43-46). Thus far, it has
hardly been explored whether arsenite can affect SP-1 and AP-2
activity. Additional experiments should be performed to establish
whether arsenite induces functional HIF-1 and whether HIF-1 contributes
to arsenite-induced VEGF expression.
In both OVCAR-3 and H134 cells, HIF-1 mRNA levels remained
unaltered upon arsenite treatment, indicating that induction of HIF-1 protein is regulated on the protein level. Many activators of
HIF-1, including hypoxia, have been shown to induce HIF-1 protein
expression by inhibiting ubiquitination and degradation (15). To
investigate whether arsenite induces HIF-1 protein accumulation
through a similar mechanism in OVCAR-3 and H134 cells, we assessed the
kinetics of the decay of arsenite-induced HIF-1 protein in the
absence of protein synthesis. As a reference for the stability of
HIF-1 under normoxia in nontreated OVCAR-3 and H134 cells, we
investigated the decay of hypoxia-stabilized HIF-1 protein upon
reoxygenation in the same manner. The decay of arsenite-induced HIF-1 protein was found to be significantly slower in comparison with the very rapid decay of hypoxia-stabilized HIF-1 protein observed upon reoxygenation. In agreement with findings in other cell
types (12, 13, 35), these results confirm that HIF-1 protein is
unstable in normoxic OVCAR-3 and H134 cells and suggest that arsenite
induces the accumulation of HIF-1 protein by inhibiting its degradation.
As increased ROS production has been suggested to mediate the
stabilization of HIF-1 (11, 19, 20), we assessed the role of ROS in
arsenite-induced HIF-1 protein and VEGF expression. By using
DCFH-DA, we demonstrated that arsenite increased the production of
hydrogen peroxide in a time-dependent manner in OVCAR-3
cells. Pretreatment with catalase or with the hydroxyl radical
scavenger mannitol efficiently attenuated arsenite-induced hydrogen
peroxide production, but failed to suppress elevation of HIF-1
protein and VEGF mRNA levels. These results strongly suggest that
both effects of arsenite are independent of increased ROS production.
The role of ROS in the regulation of HIF-1 and/or VEGF is
controversial. By investigating cells lacking mitochondrial DNA, Chandel et al. (20) have provided evidence that increased
production of mitochondrial ROS is required for stabilization of
HIF-1 protein in response to hypoxia. In a recent study in two
different cell types that were also depleted of mitochondrial DNA,
HIF-1 activation by hypoxia was unaffected (47). Another model proposes
that the response to hypoxia may be caused by a decreased production of
ROS by NADPH oxidase (18). In this model, decreased production of ROS is postulated to have an inhibitory effect on HIF-1 protein degradation. In line with this hypothesis, Haddad et al.
(48) have demonstrated that antioxidants prevent the decay of HIF-1 protein upon reoxygenation. The role of ROS in the regulation of
HIF-1 and VEGF in normoxic cells is also not well established. Results from Chandel et al. (19) have indicated that
HIF-1 protein stabilization and HIF-1-dependent
transcription in response to the transition metal cobalt (cobalt
chloride) were mediated by increased production of cytoplasmic ROS. In
contrast, inhibition of cobalt chloride-induced ROS production was
found not to influence HIF-1-dependent transcription in a
study of Salnikow et al. (49). The reason for these
controversial results is not clear, but may be attributed to
differences between the investigated cell types. In fact, ROS-mediated
induction of HIF-1 by growth factors and hormones under normoxia also
seems to be a cell type-specific effect (11). Thus, some reports,
including our own results obtained with arsenite, suggest that elevated
levels of ROS do not trigger HIF-1 activation per se.
Part of the biological effects of arsenite are likely to be caused by
its ability to lower intracellular GSH levels and by its binding to
free thiol (SH) groups of critical cellular proteins (28).
Consistently, elevation of GSH levels is found to antagonize the
effects of arsenite (36, 37). In agreement with these studies, we
demonstrated that intracellular GSH levels also modulate the effects of
arsenite on HIF-1 protein and VEGF expression in OVCAR-3 cells.
Pretreatment with GSH or NAC (a precursor for GSH) completely
attenuated induction of HIF-1 protein and VEGF mRNA levels,
whereas upon depletion of intracellular GSH by BSO the reverse was
shown. Thus, elevated levels of intracellular GSH inhibit
arsenite-induced HIF-1 protein and VEGF mRNA levels.
The exact mechanism by which intracellular GSH antagonizes induction of
HIF-1 protein and VEGF expression remains to be determined. It is
unlikely that induction of HIF-1 protein and VEGF expression by
arsenite is simply mediated by GSH depletion. As demonstrated, suppression of intracellular GSH by BSO pretreatment did not influence HIF-1 protein and VEGF mRNA levels in OVCAR-3 cells. It is more conceivable that induction of VEGF expression and HIF-1 protein by
arsenite involves its binding to thiol groups of cellular proteins. GSH
may antagonize both effects of arsenite by competitive interference with the interaction between arsenite and the thiol groups of cellular
proteins (29, 30). High levels of GSH may thus prevent arsenite from
binding to these proteins.
It is of interest to identify the cellular proteins whose interaction
with arsenite results in the induction of HIF-1 protein and VEGF
expression. A possible candidate may be the HIF-1 protein itself.
The degradation of HIF-1 under normoxic conditions is thought to be
targeted by the tumor suppressor protein von Hipple Lindau (pVHL) (50,
51). pVHL is part of a multiprotein complex possessing associated E3
ubiquitin-ligase activity (51). In normoxic cells, HIF-1 protein
exists in a complex with VHL. In hypoxic cells and in normoxic cells
treated with cobalt chloride or with the iron chelator desferrioxamine,
the VHL·HIF-1 protein complex is dissociated, which
prevents the degradation of HIF-1 protein (50, 52, 53). The
interaction of HIF-1 with pVHL is mediated by a small domain in the
HIF-1 protein called the oxygen-dependent-degradation
domain (50). This domain contains an unpaired cysteine residue (54).
Although arsenite is thought to have a much higher affinity for
dithiols (vicinal thiols) than for monothiols (28), it is possible that
arsenite disrupts the HIF-1·VHL protein complex by binding to the
thiol group of this cysteine residue.
Other potential targets for arsenite may be some of the enzymes that
participate in the ubiquitin-proteasome system. Arsenite has been
suggested to inhibit ubiquitin-dependent protein
degradation at multiple steps through its binding with vicinal thiol
groups of different components of this proteolytic pathway (55).
Although this effect of arsenite has only been demonstrated in rabbit
reticulocytes and in reticulocyte lysates (55), it cannot be excluded
that arsenite induces HIF-1 protein accumulation by acting as a
general inhibitor of the ubiquitin-proteasome system. General
proteasome inhibitors have indeed been shown to induce HIF-1 protein
accumulation under normoxic conditions. These agents appear, however,
incapable of inducing functional HIF-1 because they were found not to
stimulate HIF-1-dependent transcription (14). The fact that
arsenite induces VEGF expression may indicate that arsenite influences
additional steps in the regulation of HIF-1 or that this agent
regulates HIF-1 protein through a different mechanism.
Another mediator of arsenite-induced HIF-1 protein stabilization and
VEGF expression may be nitric oxide, the production of which is
increased in some cell types upon arsenite treatment (56).
Interestingly, nitric oxide also reacts with thiol groups of cellular
proteins, and nitrosylation of thiol residues has been suggested to
play a role in nitric oxide-induced stabilization of HIF-1 protein
under normoxic conditions (57). Inhibitors of cellular enzymes that
synthesize nitric oxide (nitric-oxide synthase inhibitors) do,
however, not significantly affect arsenite-induced HIF-1 protein
accumulation and VEGF expression in OVCAR-3 cells (data not shown).
In addition to ROS and pVHL, the one or more signaling pathways that
regulate HIF-1 activity contain several other intermediates. Inhibitors
of phosphatidylinositol 3-kinase and of the small GTPase Rac-1 can
block HIF-1 protein stabilization and transactivation by hypoxia.
Inhibition of the mitogen-activated protein kinase (MAPK) family
members p38 MAPK and p44/p42 MAPKs also has a negative effect on
the transactivation function of HIF-1 in hypoxic cells (58). These
factors have been implicated in the regulation of HIF-1 and/or
VEGF expression in normoxic cells as well (58-61). Interestingly, arsenite can enhance the activity of
phosphatidylinositol 3-kinase as well as of Rac1, p38 MAPK, and p44/p42
MAPKs (37, 62, 63). We are currently assessing a possible involvement of these components in the effects of arsenite on HIF-1 protein and
VEGF expression.
It is interesting to note that our results with arsenite may also be of
clinical relevance. Despite the fact that trivalent arsenic
(As3+) is toxic to humans, arsenic trioxide
(As2O3) appears to be a promising agent in the
treatment of cancer when used at low dosages (24).
As2O3 has been shown to promote apoptosis in
endothelial cells and in several types of tumor cells. Based on the
observation that low dosages of As2O3
(~10
6 M) have a significant cytotoxic
effect on human ovarian cancer cell lines, it was suggested that
As2O3 may also be a useful agent for the
treatment of ovarian cancer (64). Although the effects of arsenite on
VEGF expression and HIF-1 protein in OVCAR-3 and H134 cells were
observed at relatively high concentrations (30 and 100 µM), our study may indicate that caution should be taken with regard to such an approach.
In this study, we show for the first time that HIF-1 protein and
VEGF expression can be induced by a thiol-reactive agent in human
ovarian cancer cells. These findings may have implications for
cytotoxic agents in cancer treatment, such as highly reactive platinum
compounds and alkylating agents, which are susceptible of interacting
with thiol groups of cellular sulfhydryls.
Protein by the Oxidative Stressor
Arsenite*
,
TOP
ABSTRACT
INTRODUCTION
To whom correspondence should be addressed. Tel.: 31-20-444-8327;
Fax: 31-20-444-4355; E-mail: mca.duyndam.oncol@med.vu.nl.
